Enhanced production of taxol and taxanes by cell cultures of taxus species

ABSTRACT

This invention provides methods whereby taxol, baccatin III, and other taxol-like compounds, or taxanes, can be produced in very high yield from all known  Taxus  species, e.g.,  brevifolia, canadensis, cuspidata, baccata, globosa, floridana, wallichiana, media  and  chinensis . Particular modifications of culture conditions (i.e., media composition and operating modes) have been discovered to enhance the yield of various taxanes from cell culture of all species of  Taxus . Particularly preferred enhancement agents include silver ion or complex, jasmonic acid (especially the methyl ester), auxin-related growth regulators, and inhibitors of the phenylpropanoid pathway, such as 3,4-methylenedioxy-6-nitrocinnamic acid. These enhancement agents may be used alone or in combination with one another or other yield-enhancing conditions. While the yield of taxanes from plant cell culture of  T. chinensis  is particularly enhanced by use of one or more of these conditions, yield of taxanes for all  Taxus  species has been found to benefit from use of these conditions.

This application is a continuation-in-part of International applicationPCT/US97/08907, designating the U.S., filed May 27, 1997, and acontinuation-in-part of U.S. Ser. No. 08/653,036, filed May 24, 1996,which is a continuation-in-part of U.S. Ser. No. 08/370,494, filed Jan.9, 1995, which is a divisional of U.S. Ser. No. 07/874,344, now U.S.Pat. No. 5,407,816, filed Apr. 24, 1992, which is a continuation-in-partof U.S. Ser. No. 07/839,144, filed Feb. 20, 1992. The text of eachpriority application is expressly incorporated herein by reference tothe extent that the text of the respective priority application differsfrom this application.

BACKGROUND OF THE INVENTION

A. Field of the Invention

This invention is directed to methods for the enhanced production andrecovery of taxol, baccatin III and other taxanes by cell cultures ofTaxus species.

B. Related Art

The Taxane Supply Challenge

Taxol is a diterpenoid alkaloid originally isolated from the bark of thepacific yew, Taxus brevifolia (Wani, et al. 1971, J. Am. Chem. Soc., 93,2325-2327). Interest in taxol began when the National Cancer Institute(NCl), in a large-scale screening program, found that crude barkextracts exhibited anti-tumor activities. Since then, clinical trialshave confirmed that taxol is extremely effective against refractoryovarian cancers, and against breast and other cancers. Taxol has beenpronounced as a breakthrough in chemotherapy because of itsfundamentally different mechanism of cytotoxicity, i.e., by inhibitingdepolymerization of microtubules (see Rowinsky, et al., 1990, J. Natl.Cancer Inst., 82, 1247-1259).

A daunting variable in the taxol equation has been supply. Bark-derivedtaxol has been discontinued as the primary source of commercial drug;large-scale production has been achieved by semi-synthesis, i.e.,chemical attachment of a side chain to the plant-derived precursor,10-deacetylbaccatin III. Total synthesis, while accomplished by academiclaboratories, shows little promise as a viable commercial route totaxol. There is therefore an urgent need to develop cost-effective,environmentally-benign, and consistent sources of supply to keep up withthe growing demand for taxol.

In addition to taxol, there is an urgent need to develop processes forthe commercial production of related taxane molecules. Derivatives oftaxol such as Taxotere have already been introduced into the worldmarket. Further, tremendous research activity is being focused on thediscovery and development of novel taxane derivatives with advantageousactivity. These advances are likely to create an ongoing need for largequantities of an appropriate starting “skeleton” molecule from which anygiven derivative could be effectively synthesized.

One example of such a molecule is the aforementioned precursor,10-deacetylbaccatin III, which is used as the starting point forsemi-synthetic taxol. Another desirable starting molecule forsemi-synthetic production of taxol and other derivatives is baccatinIII. Baccatin III is normally not accumulated as a major taxane inplanta, and hence there is no facile large-scale natural source for thismolecule. However, it is a very desirable starting point forsemi-synthesis because of its chemical closeness to taxol; for example,the steps that are required for acetylation of the 10 position of10-deacetylbaccatin III are circumvented if baccatin III is the startingpoint rather than 10-deacetylbaccatin III.

This invention is related to the development of plant cell culture-basedprocesses for the commercial production of taxol, baccatin III and othertaxanes.

Tissue Cultures as a Source of Plant-Derived Chemicals

The ability of plant cells to divide, grow, and produce secondarymetabolites under a variety of different cultural regimes has been amplydemonstrated by a number of groups. At present, two compounds, shikonin(a red dye and anti-inflammatory) and ginsengoside (a tonic in orientalmedicine) are produced by tissue-culture processes in Japan. Many otherprocesses are reportedly close to commercialization, including vanillin,berberine and rosmarinic acid (see Payne, et al. 1991, “Plant Cell andTissue Culture in Liquid Systems,” Hanser Publishers, Munich).

The advantages of a plant cell culture process for taxol, baccatin III,and taxanes are many: (i) A cell culture process ensures a limitless,continuous and uniform supply of product, and is not subject to pests,disasters and seasonal fluctuations, (ii) cell cultures can becultivated in large bioreactors, and can be induced to overproduce thecompound of interest by manipulating environmental conditions, (iii)cell cultures produce a simpler spectrum of compounds compared to barkor needles, considerably simplifying separation and purification, (iv) acell culture process can adapt quickly to rapid changes in demand betterthan agriculture-based processes, (v) besides supplying taxol, baccatinIII or other precursors, a cell culture process could also producetaxane compounds that exhibit advantageous bioactivity profiles, or thatcould be converted into other bioactive derivatives.

Since aseptic, large-scale, plant cell cultivation is inherentlyexpensive, a cell culture process becomes commercially relevant onlywhen these costs are offset by high productivity. Every plant speciesand target metabolite is different, and different approaches arenecessary for every particular system. This invention focuses oncreative and skilled approaches for obtaining highly productive plantcell cultures for taxol, baccatin III, and taxane production.

Problems with Tissue Cultures of Woody Plants and Conifers

A historical survey of the literature suggests that whereas herbaceousplants have been relatively easily manipulated in culture, productivecultures of woody plants and conifers have been achieved only withdifficulty.

The growth of secondary metabolite producing gymnosperm- andconifer-cultures have been generally low. For example, Berlin and Witte,(1988, Phytochemistry, 27, 127-132) found that cultures of Thujaoccidentalis increased their biomass by only ca. 30% in 18 days. VanUden et al. (1990, Plant Cell Reports, 9, 257-260) reported a biomassincrease of 20-50% in 21 days for suspensions of Callitris drummondii.Westgate et al. (1991, Appl. Microbiol. Biotechnol., 34, 798-803)reported a doubling time of ca. 10 days for suspensions of thegymnosperm, Cephalotaxus harringtonia. As summarized by Bornman (1983,Physiol. Plant. 57, 5-16), a tremendous amount of effort has beendirected towards medium development for spruce suspensions (Piceaabies). This collective work demonstrates that gymnosperm suspensionsare indeed capable of rapid growth, but that no generalities can beapplied, and that media formulations for different cell lines must beoptimized independently.

A survey of secondary metabolite productivity among gymnosperm culturesalso points to the difficulty of inducing rapid biosynthesis compared toherbaceous species. For example, cultures of Cephalotaxus harringtoniaproduced terpene alkaloids at a level of only 1% to 3% of that found inthe parent plant (Delfel and Rothfus, 1977, Phytochemistry, 16,1595-1598). Even upon successful elicitation, Heinstein (1985, Journalof Natural Products, 48, 1-9) was only able to approach the levelsproduced in the parent plant (ca. 0.04% dry weight total alkaloids). VanUden et al (1990) were able to induce suspension cultures of the coniferCallitris drummondii to produce podophyllotoxin, but only at levels onetenth of that produced by the needles. The ability of Thuja occidentalisto produce significant levels of monoterpenes (10-20 mg/L) and thediterpenoid dehydroferruginol (2-8 mg/L) has been convincinglydemonstrated by Berlin et al. (1988). However, these results wereobtained with a slow-growing (30% biomass increase in 18 days) and lowcell density (5 to 7 grams dry weight per liter) culture.

Cell Culture for Taxane Production

The difficulties in achieving rapid growth and high productivityencountered in gymnosperm-suspensions have generally been reflected inthe reports so far on taxane production in Taxus cell cultures.

Jaziri et al. (1991, J Pharm. Belg., 46, 93-99) recently initiatedcallus cultures of Taxus baccata, but were unable to detect any taxolusing their immunosorbent assay. Wickremesinhe and Arteca (1991, PlantPhysiol., 96, (Supplement) p. 97) reported the presence of 0.009% thyweight taxol in callus cultures of Taxus media (cv. hicksii), butdetails on the doubling times, cell densities, and the time-scale overwhich the reported taxol was produced, were not indicated.

U.S. Pat. No. 5,019,504 (Christen et al. 1991) describes the productionand recovery of taxane and taxane-like compounds by cell cultures ofTaxus brevifolia. These workers reported taxol production at a level of1 to 3 mg/L in a two- to four-week time frame. They also reported a cellmass increase of “5-10 times in 3-4 weeks”, which corresponds todoubling times of ca. 7 to 12 days.

Significant increases in taxane titers and volumetric productivity arerequired before an economically-viable plant cell culture process fortaxane production can supply the projected annual demand of manyhundreds of kilograms per year.

SUMMARY OF THE INVENTION

The objects of this invention include the formulation of specialenvironmental conditions to foster rapid growth, high cell densities,and high cell viabilities. (The growth characteristics reported in thisstudy surpass previous results by a significant factor.)

An object of this invention is to produce taxanes at high rates bycareful selection of cell lines, careful choice and manipulation ofmedium conditions, incorporation of enhancement agents, and carefulselection of process-operating modes.

The objects of this invention include the ability to manipulate theprofile of taxanes produced by altering media formulations andenvironmental conditions. In particular, it is an object to encouragecells to produce taxol or baccatin III as the predominant taxaneproduct, and/or to suppress the production of the by-productcephalomannine, thereby providing an elegant biological solution to anexpensive and important downstream separation and purification problem.These and other objects are met by one or more of the embodiments ofthis invention.

The inventors have discovered that taxol, baccatin III, and othertaxol-like compounds, or taxanes, can be produced in very high yieldfrom all known Taxus species, e.g., brevifolia, canadensis, cuspidata,baccata, globosa, floridana, wallichiana, media and chinensis. Further,by the methods of this invention it is possible to obtain taxol,baccatin III, and other taxanes in a much shorter time frame thanpreviously reported. In particular, the inventors found that thespecies, Taxus chinensis, is capable of rapid growth and of producingextremely high levels of taxol, baccatin III, and taxanes within a shortperiod of time. With the species Taxus chinensis, the inventors havebeen able to manipulate cells to yield taxol, baccatin III, and taxanesin amounts far in excess of the amounts obtained from tissue cultures ofthe other Taxus species.

Particular modifications of culture conditions (i.e., media compositionand operating modes) have been discovered to enhance the yield ofvarious taxanes from cell culture of all species of Taxus. Particularlypreferred enhancement agents include silver ion or complex, jasmonicacid (especially the methyl ester), auxin-related growth regulators, andinhibitors of the phenylpropanoid pathway, such as3,4-methylenedioxy-6-nitrocinnamic acid. These enhancement agents may beused alone or in combination with one another or other yield-enhancingconditions. While the yield of taxanes from plant cell culture of T.chinensis is particularly enhanced by use of one or more of theseconditions, yield of taxanes for all Taxus species has been found tobenefit from use of these conditions.

In one embodiment, this invention provides a method for producingtaxanes in high yields in cell culture of a Taxus species comprisingcultivating cells of a Taxus species in suspension culture in one ormore nutrient media under growth and product formation conditions, andrecovering one or more taxanes from said cells or said medium of saidcell culture, or both, the cells being derived from callus or suspensioncultures and the nutrient media containing an inhibitor ofphenylpropanoid metabolism. Suitable inhibitors of phenylpropanoidmetabolism include 3,4-methylenedioxy-6-nitrocinnamic acid,3,4-methylenedioxycinnamic acid, 3,4-methylenedioxy-phenylpropionicacid, 3,4-methylenedioxyphenylacetic acid, 3,4-methylenedioxybenzoicacid, 3,4-trans-dimethoxycinnamic acid, 4-hydroxycinnamic acid,phenylpropiolic acid, fluorophenylalanine, 1-aminobenzotriazole,2-hydroxy-4,6-dimethoxybenzoic acid, SKF-525A, ammonium oxalate,vinylimidazole, diethyldithiocarbamic acid, and sinapic acid.

In a preferred embodiment, at least one of the one or more nutrientmedia used in the method of this invention also comprises anotherenhancement agent which may be an inhibitor of ethylene action; jasmonicacid or an ester of jasmonic acid; or an auxin-related growth regulator.In particularly preferred embodiments, the other enhancement agent is aninhibitor of ethylene action which is a silver-containing compound, or asilver complex, or a silver ion. In another particularly preferredembodiment, the other enhancement agent is jasmonic acid or an alkylester thereof, and more preferably, the alkyl group esterified tojasmonic acid has from one to six carbon atoms. In an even morepreferred embodiment, the enhancement agent is jasmonic acid or an alkylester thereof, and the medium also contains a silver-containingcompound, a silver complex or silver ion. In yet another particularlypreferred embodiment, the other enhancement agent is an auxin-relatedgrowth regulator, such as indoleacetic acid, picloram,α-naphthaleneacetic acid, indolebutyric acid, 2,4-dichlorophenoxyaceticacid, 3,7-dichloro-8-quinolinecarboxylic acid, or 3,6-dichloro-o-anisicacid.

In another embodiment, this invention provides a method for producingtaxanes in high yields in cell culture of a Taxus species by cultivatingcells of a Taxus species in suspension culture in one or more nutrientmedia under growth and product formation conditions, and recovering oneor more taxanes from said cells or said medium of said cell culture, orboth, the cells being derived from callus or suspension cultures and thenutrient media containing silver at a concentration of 900 μM or less inthe form of a silver-containing compound, or a silver complex, or asilver ion, along with at least one enhancement agent which may bejasmonic acid or an ester of jasmonic acid an auxin-related growthregulator. In a preferred embodiment, the enhancement agent is jasmonicacid or an ester of jasmonic acid, and the molar ratio of silver toenhancement agent is less than 9.5. In another preferred embodiment, theenhancement agent is an auxin-related growth regulator, and the molarratio of silver to enhancement agent is at least 0.011.

In any of the above embodiments, the one or more nutrient media may alsoinclude a taxane precursor, which may be α-phenylalanine,β-phenylalanine, or a mixture thereof. In any of the above embodiments,the one or more nutrient media may also include glutamine, glutamicacid, aspartic acid or a mixture of these amino acids, or one or morenutrient media used in cultivation of the cells may include maltose,sucrose, glucose and/or fructose as a carbon source, preferably as theprimary carbon source. In one embodiment, the nutrient medium is thesame for cell culture growth and for taxol and taxane production. In analternative embodiment, production of one or more taxanes is induced inthe culture by changing the composition of the nutrient medium. In apreferred embodiment, the medium in the culture is periodicallyexchanged, and typically the medium exchange accomplishes periodicremoval of taxanes from the culture. Preferably, cells of said Taxusspecies are cultivated by a fed-batch process.

Typically, taxol or baccatin III and/or other taxanes are recovered fromsaid cells or said medium of said cell culture, or both. Generally,cultivation of Taxus species according to this invention provides anaverage volumetric productivity of taxanes which is at least 15 mg/L/dayaveraged over the period of taxane production. The average volumetricproductivity of taxol is typically at least 10 mg/L/day computed for theperiod of taxol production. The average volumetric productivity ofbaccatin III is typically at least 15 mg/L/day computed for the periodof taxane production.

Preferably, cells cultured according to the method of this invention arecells of Taxus species, and the species may be T. brevifolia, T.canadensis, T. chinensis, T. cuspidata, T. baccata, T. globosa, T.floridana, T. wallichiana, or T. media. Preferably, the cells of a Taxusspecies used in the method of this invention are cells which producetaxol above background by ELISA in callus culture or suspension culturein medium that contains no enhancement agents. More preferably, thecells of a Taxus species used in the method of this invention are cellswhich produce taxanes in suspension culture at an average volumetricproductivity of 10 mg/L in a medium containing silver thiosulfate,methyl jasmonate and auxin.

DESCRIPTION OF THE FIGURES

FIG. 1. Biomass increase in a Taxus chinensis suspension culture lineK-1 over a typical batch growth cycle in Medium A. Error bars representthe standard deviation measured from duplicate flasks.

FIG. 2. Effect of medium exchange on days 9 and 12 on taxol (A) andtotal taxane (B) productivity in a 15-day experiment. The numbers ineach box represent the time interval (days) over which the product wasproduced. The darkened portion of the intracellular boxes represents thetaxol or total taxanes that were present in the cell inoculum at thestart of the experiment. All treatments were performed in duplicate.Taxus chinensis suspension cell line K-1 was used with Medium A aselaborated in Table 2.

FIG. 3. Spectral characteristics of a Standard Gro-Lux lamp (GTESylvania, Danvers, Mass.) used in Example 7.3.

FIG. 4. Taxane production in Taxus chinensis cell suspension K-1. Theportion of the chromatogram from 10 to 40 minutes is shown. Diode arrayscans of selected taxane peaks show a characteristic taxane UVabsorption spectrum, with a peak at 227 nm.

FIG. 5. Taxol and taxane production after prolonged cultivation inMedium C by Taxus chinensis cell line K-1. The upper panel tabulates thedata for the known and unknown taxanes, whereas the lower panel showsincremental taxol and taxane production in the 25 to 42 day time period.

FIG. 6. MS/MS confirmation of taxol in cell culture supernatant. Panel Ashows the ion spray APCI mass spectrum of authentic taxol and panel Bshows the daughter ion spectrum of the parent peak (m/z 871=taxol+NH4+).Panel C represents the ion spray APCI spectrum from a crude cell cultureextract and shows m/z 854 and 871 characteristic of taxol. Panel D showsthe corresponding daughter spectrum of m/z 871 and provides unequivocalevidence for the presence of taxol in cell culture supernatant.

DETAILED DESCRIPTION OF THE INVENTION

Plants have long provided important sources of pharmaceuticals andspecialty chemicals. These products have typically been obtained throughextraction of the harvested plant materials or by chemical synthesis.Taxol and taxanes have become one of the most important class ofanticancer agents to recently emerge from the screening of naturalproducts.

As used herein, the terms taxol-like compounds, or taxanes, are usedinterchangeably to describe a diterpenoid compound with a taxane ring.The taxanes may themselves possess antineoplastic activity, or may bemodified to yield bioactive compounds. The term total taxanes refers toall taxanes that exhibit a characteristic UV absorbance as described inExample 5 below.

As used herein, the term “callus” is used to describe a mass of culturedplant cells that is structurally undifferentiated, and is cultivated onsolidified medium. As used herein, the term “suspension culture” is usedto describe structurally undifferentiated cells that are dispersed in aliquid nutrient medium. It is understood that suspension culturescomprise cells in various stages of aggregation. A range of aggregatesizes are encountered in the suspensions described in this invention,with sizes ranging from tens of microns in diameter (single cells orfew-aggregated cells) to aggregates many millimeters in diameter,consisting of many thousands of cells.

The plant material useful in this invention may be obtained from anyknown Taxus species, e.g., brevifolia, canadensis, cuspidata, baccata,globosa, floridana, wallichiana (also referred to as yunnanensis),media, fastigiata and chinensis (including the synonymous species, suchas sumatrama, celebica, and speciosa, and the subspecies chinensis var.mairei). In particular, the inventors have identified the species Taxuschinensis as capable of producing significant quantities of taxol,baccatin III, and taxanes at high volumetric productivities.

It has been found by the inventors that specific taxane content varieswith plant species, and within plant species from tissue source andspecific trees. Selecting a high yielding source and culture for taxaneproduction is an important first step towards providing sufficientquantities of taxanes for therapeutic use.

Benchmarks for Commercial Relevance

A number of benchmarks may be used to gauge the commercialattractiveness and viability of a given plant-cell-culture-based processfor taxane production. The benchmarks should characterize and underpinthe key performance parameters of the process, including fermentationcosts, the ease of downstream recovery, and the capacity of production.The benchmarks that will be described here are the broth titer and thevolumetric productivity.

The broth titer is defined as the concentration of product in the wholebroth, and is usually expressed as milligrams of product per liter ofbroth (mg/L). By definition, the whole broth titer does not distinguishbetween the intracellular and extracellular portions of the product. Thebroth titer is typically used to characterize the performance of a batchor fed-batch process. A higher broth titer implies a greater productioncapacity for a given reactor volume, and concomitantly, lower unitproduction costs. Similarly, a high-titer product is usually easier torecover in high yield, thus leading to further improvements in unitproduction costs.

The volumetric productivity is defined as the amount of product producedper unit reaction volume per unit time, and is commonly expressed inunits of milligrams per liter per day. For the purposes of taxaneproduction, the time scale is defined as the time frame during whichproduction takes place at the production scale immediately precedingharvest and recovery. The volumetric productivity complements the titeras a benchmark for batch and fed-batch processes, and is particularlyuseful for characterizing processes where the product is removed duringproduction, for example, by periodic medium exchange or another methodof removal. A high volumetric productivity implies greater productioncapacity for a given reactor volume over a give time period, andconcomitantly, lower unit production costs and greater overall processperformance.

In certain cases the volumetric productivity is used to gauge theintrinsic capability of a biological process—for example, in the earlierstages of process development, it is useful to measure the productivityover the most productive part of the production cycle, i.e., over ashort time period when the rates of biosynthesis are at their highest.This is typically referred to as the maximal instantaneous volumetricproductivity. However, in gauging the performance of a process, the moreappropriate benchmark is the average volumetric productivity in whichthe productivity is measured over the entire productive phase. Clearly,in order to achieve the highest average volumetric productivity, themaximal instantaneous productivity must be maintained through themajority of the productive phase. Unless otherwise qualified, the termvolumetric productivity refers to the average volumetric productivity,determined for the entire production phase Typically, production phaseis initiated by changes in nutrient medium composition, either byreplacing growth medium with production medium or by adding enhancementagents which induce a significant enhancement in taxane production.

Initiation of Taxus Cell Lines

Taxus plant material may be collected from all over North America aswell as from other continents. The culture is initiated by selectingappropriate Taxus tissue for growth. Tissue from any part of the plant,including the bark, cambium, needles, stems, seeds, cones, and roots,may be selected for inducing callus. However, for optimum yield oftaxol, needles and meristematic regions of plant parts are preferred.Most preferred are new growth needles (e.g., one to three months old),which can generally be identified by a lighter green color. The term“new growth” is broadly intended to mean plant needle production withinthat year's growing season.

To prevent contamination of the culture, the tissue should besurface-sterilized prior to introducing it to the culture medium. Anyconventional sterilization technique, such as CLOROX (a trademark ownedby the Clorox Company for bleach) treatment would be effective. Inaddition, antimicrobial agents such as cefoxitin, benlate, cloxacillin,ampicillin, gentamycin sulfate, and phosphomycin may be used for surfacesterilization of plant material.

Callus Growth

Cultures will typically exhibit variability in growth morphology,productivity, product profiles, and other characteristics. Sinceindividual cell lines vary in their preferences for growth mediumconstituents, many different growth media may be used for induction andproliferation of the callus.

The appropriate medium composition varies with the species beingcultured. The preferred media for the different species are listed inTable 3. For example, although others may be used, the preferred growthnutrient media for Taxus chinensis are A, D, I, J, K, L, M, O, P. Thesemedia preferably contain the ingredients listed in Table 2. Cultures arepreferably carried out with medium components incorporated at the levelsshown in Table 2, although the skilled artisan will recognize that somevariation in these levels will not adversely affect cell growth. Forexample, when medium A is used, growth hormones or regulatorsincorporated into the medium in an amount between 1 ppb to 10 ppm, andpreferably at 2 ppb to 1 ppm. When medium D is used, the growth hormonesor regulators are incorporated at levels ranging from 1 ppb to 10 ppm,and preferably at 2 ppb to 2 ppm. The amounts of other mediumingredients can be incorporated at levels ranging from 1/10thconcentration to three times the concentrations indicated in Table 2.

Production of taxanes in large quantities is facilitated by cultivatingTaxus cells in suspension culture. Generally, suspension culture can beinitiated using a culture medium that was successful in callus culture.However, the requirements for suspension culture, and particularly forhighly efficient production of taxanes, may be better met bymodification of the medium. It has been found that when Taxus cells arecultured in modified culture medium and processing parameters tailoredaccording to the method of this invention, the yield of one or moretaxanes from the culture is substantially increased.

As used herein, the term “nutrient medium” is used to describe a mediumthat is suitable for the cultivation of plant cell callus and suspensioncultures. The term “nutrient medium” is general and encompasses both“growth medium” and “production medium”. The term “growth medium” isused to describe an nutrient medium that favors rapid growth of culturedcells. The term “production medium” refers to an nutrient medium thatfavors taxol, baccatin III, and taxane biosynthesis in cultured cells.It is understood that growth can occur in a production medium, and thatproduction can take place in a growth medium; and that both optimumgrowth and production can take place in a single nutrient medium.

Suspension Growth

Taxus suspension cultures are capable of rapid growth rates and highcell densities like other plant cell cultures. However, optimalconditions may vary from one cell line to another, and accordingly,methods leading towards rapid optimization for any given cell line mustbe considered.

The cultures of various Taxus species are cultivated by transfer intonutrient media containing macro- and micro-nutrient salts, carbonsources, nitrogen sources, vitamins, organic acids, and natural andsynthetic plant growth regulators. In particular, nutrient medium forsuspension culture of Taxus cells will typically contain inorganic saltsthat supply the macronutrients calcium, magnesium, sodium, potassium,phosphate, sulfate, chloride, nitrate, and ammonium, and themicronutrients such as copper, iron, manganese, molybdenum, zinc, boron,cobalt, iodine, and nickel. The medium will also typically containvitamins such as myo-inositol, thiamine, ascorbic acid, nicotinic acid,folic acid, pyridoxine and optionally biotin, pantothenate, niacin andthe like. These components may be present at concentration ranges of1/30th to thirty times the concentrations listed in Table 2, andpreferably at 1/20th to twenty times the concentrations listed in Table2, more preferably at ⅓ to three times the concentrations listed inTable 2, and most preferably at the concentrations listed in Table 2.

The nutrient medium will also contain one or more carbon sources, andwill typically contain a primary carbon source, which is defined as asource that provides over 50% of the total carbon in the nutrientmedium. The primary carbon source is preferably lactose, galactose,raffinose, mannose, cellobiose, arabinose, xylose, sorbitol, orpreferably glucose, fructose, sucrose or maltose. The concentration ofthe primary carbon source may range from 0.05% (w/v) to 10% (w/v), andpreferably from 0.1% (w/v) to 8% (w/v).

The nutrient medium will also contain a nitrogen source, which, inaddition to any nitrogen added in the form of macronutrient salts, willpreferably be provided at least in part by an organic nitrogen source(e.g., one or more amino acids such as glutamine, glutamic acid, andaspartic acid, or protein hydrolyzates). These organic nitrogen sourcesmay supply nitrogen at concentrations ranging from 0.1 mM to 60 mM, andpreferably from 1 to 30 mM. The medium may also contain one or moreorganic acids such as acetate, pyruvate, citrate, oxoglutarate,succinate, fumarate, malate, and the like. These components may beincluded in the medium at concentrations of 0.1 mM to 30 mM, andpreferably at concentrations of 0.5 mM to 20 mM.

The medium will also typically contain one or more natural or syntheticplant growth regulators, including auxin-related growth regulators suchas picloram, indoleacetic acid, 1-naphthaleneacetic acid, indolebutyricacid, 2,4-dichlorophenoxyacetic acid, 3,7-dichloro-8-quinolinecarboxylicacid, 3,6-dichloro-o-anisic acid, and the like, cytokinin-related growthregulators such as N⁶-benzyladenine, 6-[γ,γ-dimethylallylamino]purine,kinetin, zeatin, N-phenyl-N′-1,2,3-thidiazol-5-ylurea (thidiazuron) andrelated phenylurea derivatives and the like, gibberrellins such as GA₃,GA₄, GA₇, and GA derivatives, abscisic acid and its derivatives,brassinosteroids, and ethylene-related growth regulators. Additionalsuitable auxin-related plant growth regulators are listed below. Itshould be noted that the nutrient medium may contain more than onegrowth regulator belonging to a single class, for example, more than asingle auxin-related regulator, or more than one cytokinin-relatedregulator. The growth regulators will be preferably incorporated intothe medium at a concentration between 10⁻¹° M to 10⁻³ M, preferably at10 to 3×10⁻⁵ M, and more preferably at the concentrations listed inTable 2.

Unless otherwise indicated, growth media as defined herein provide asuitable starting point for routine optimization of callus culture mediaand production media. It is a routine matter for those skilled in theart to incorporate, modify, and manipulate particular classes ofcomponents, and components from within a given class, to achieve optimumperformance; particular media modifications are provided in the Tablesand Examples below.

The liquid cultures are exposed to a gaseous environment such as air andpreferably shaken or otherwise agitated to allow for proper mixing ofculture components. The cultures are maintained at a temperature between23° C. and 27° C., although under appropriate conditions and/orcircumstances, temperatures could range from 0° C. to 33° C. The pH maybe from about 3 to 7 and preferably between 4 to 6. The culture may begrown under light conditions ranging from total darkness to total light(narrow band and/or broad spectrum) for various periods of time.

Doubling times have been measured by monitoring time-dependent biomassincrease, as well as by simply monitoring the growth index duringroutine subculture. Maximum dry weight densities of 15-24 grams perliter have been achieved. The growth characteristics of various Taxusspecies suspensions are elaborated in Example 4.

Taxane Production Conditions

If secondary metabolite formation in a suspension culture takes placeconcurrently with growth, the metabolite is termed growth-associated,and a single medium formulation may be sufficient to achieve good growthand high level production. In many other systems, it has been found thatrapid growth and high product formation do not take place concurrently.In such cases, growth and production phases are separated and a mediumfor each phase is developed independently (reviewed in Payne et al.1991, Plant Cell and Tissue Culture in Liquid Systems, Hanserpublishers, Munich). In the case of taxane production in Taxus, growthand product formation can be separated, and independent media have beendeveloped for each.

In a preferred mode of this invention, the composition of the mediumduring the cell growth phase is different from the composition of themedium during the taxane production phase. For example, the identity andlevel of the carbon sources, particularly the primary carbon source, maychange between the growth phase and the production phase. Preferably theproduction medium will contain sugar at a level higher than that of thegrowth medium. More preferably the initial sugar level in the productionmedium may be 2-20 times higher in the production phase than the growthphase. The primary carbon source is preferably lactose, galactose,raffinose, mannose, cellobiose, arabinose, xylose, sorbitol, orpreferably glucose, fructose, sucrose or maltose. The concentration ofthe primary carbon source may range from 0.05% (w/v) to 10% (w/v), andpreferably from 0.1% (w/v) to 8% (w/v). Particularly preferred carbonsources for production of taxol or baccatin are maltose, sucrose,glucose and/or fructose. In particularly preferred embodiments, thesesugars will be incorporated in initial nutrient medium at concentrationsof at least 3.5%.

The identity and the level of organic supplements, which may include,vitamins, organic nitrogen sources such as amino acids, as well as thepresence or levels of the enhancement agents described below, may changeor may differ in the media. The identity and levels of the natural orsynthetic plant growth regulators may differ between the media.Similarly the levels and identity of macronutrient and micronutrientsalts may also differ between the growth and production media.Preferably, the salt content is reduced in the production mediumrelative to the growth medium, optionally, nitrate and sulfate salts arereduced disproportionately and more preferably the extent of reductionis a reduction by a factor of 2-20 fold. However, it is understood thata single growth/production medium may be formulated for this culture.

The production media developed here not only increase taxane formation,but also direct cellular biosynthesis towards production of particulartaxanes, such as taxol or baccatin III. In addition, production ofinterfering by-products such as cephalomannine is minimal compared tobark tissue. The production media developed here also promote prolongedcell viability and biosynthesis, and in addition, cause significantlevels of product to be secreted into the extracellular medium. Thesecharacteristics are extremely important in the operation of an efficientcommercial scale process for taxane production.

Methods for the extraction and recovery of taxol and taxanes from cellsand the medium follow conventional techniques (see, e.g., Example 5).The immuno-assay (ELISA) technique largely followed the protocolssupplied by Hawaii Biotechnology in the commercially available kit (seealso, Grothaus et al. 1995, Journal of Natural Products, 58, 1003-1014incorporated herein by reference). The antibody may be specific for anytaxane, such as taxol or baccatin III, or less specifically, for thetaxane skeleton. High performance liquid chromatography methods wereslightly modified from existing protocols as elaborated in Example 5.Under the conditions used in this invention, clear resolution of taxanepeaks was achieved, resulting in accurate detection and quantitation.Because of the possibility of co-eluting non-taxane components, thespectral purity of taxane peaks were routine by checked by diode arraybefore integration of peak areas. Retention times of taxane standardsare listed in Example 5, and a sample chromatogram is included in FIG.4.

For higher plants, light is a potent factor in secondary metabolism bothin intact plant as well as in cell cultures. Both the intensity andwavelength of light are important (Seibert and Kadkade 1980, “PlantTissue Culture as a Source of Biochemicals.” E. J. Staba (ed), CRCPress, Boca Raton, Fla., pp. 123-141). For example, flavanoid andanthocyanin biosynthesis are usually favored by high intensitycontinuous light, while dark-cultivated cultures may be preferable forother metabolites. Increase in greening or photosynthetic capacity ofcultured cells may also increase product formation or product spectrum.The inventors' studies involved the use of broad-band and well asspecific narrow-band light sources. As shown in Example 7.3., lightexposure can bring about increased taxol accumulation as well assecretion into the medium. The stimulatory effect of light on taxolproduction suggests the existence of unique control mechanisms forbiosynthesis of taxanes. The nature of the photoreceptor and biochemicalcharacteristics of light-induced stimulation are not yet clear. However,the incorporation of enhancement agents, in accordance with theteachings of this invention, render the role of light as less criticalfor optimum performance.

In addition to non-volatile dissolved nutrients, gaseous components,primarily oxygen, carbon dioxide, and ethylene (a plant hormone), playcritical roles in growth and product formation. Two parameters areimportant. The dissolved gas concentrations favoring growth and taxolformation are obviously important since they dictate reactor operatingconditions. In addition, the rates of consumption or production need tobe incorporated into reactor design, so that the optimum specifiedconcentrations can be maintained.

Besides its importance in respiration, oxygen can also dramaticallyaffect the rate of secondary metabolite biosynthesis. A high saturationconstant for an oxygen-requiring step on a secondary biosyntheticpathway may require cells to be subjected to high oxygen levels in thereactor. The importance of CO₂ supplementation in maintaining highgrowth rates has been documented. Ethylene, a plant hormone, playspleiotropic roles in all aspects of plant growth and development,including secondary metabolism (e.g., see Payne et al., 1991).

The inventors have found that certain gas concentration regimes mayfavor growth and secondary metabolism in cell cultures. For example, arange of oxygen concentrations may be compatible with culturecultivation, from 1% of air saturation to up to 200% of air saturation,and preferably in the range of 10% to 100%, and most preferably in therange of 25% to 95%. A range of carbon dioxide concentrations may becompatible with culture cultivation, from 0.03% (v/v in the gas phasethat is in equilibrium with the culture medium) to 15% (v/v), andpreferably in the range of 0.3% to 8% (v/v). The optimal concentrationsof dissolved gases may differ with respect to the cell metabolism, forexample, cells undergoing rapid growth may have different optima thancells undergoing taxane biosynthesis, which typically favor higheroxygen levels, and are less sensitive to higher carbon dioxide levels.The optima may also vary with the kinetics of the culture; for example,cells in the lag phase may prefer different dissolved gas concentrationsthan cells in the logarithmic growth phase.

Dissolved gases may interact with other culture components and with theaction of enhancement agents in many ways. For example, oxygenrequirements may change upon elicitation or stimulation of biosynthesis.Increases in respiration rates as a wound response are commonly observedwhen plant cell cultures are elicited. Elicitors or stimulators maymediate their action via ethylene, or may affect ethylene productionindependently of promoting secondary metabolism. In such cases, it maybe desirable to substitute a microbial elicitor preparation withethylene, and perhaps prevent toxicity associated with other microbialcomponents in the elicitor preparation. Alternatively, it may beadvantageous to inhibit the action of ethylene, thereby allowing theelicitor or stimulant to promote secondary metabolism in a moreexclusive, and thereby more effective, manner. As described below,silver ion, a component known to affect ethylene action, doesadvantageously modify taxane biosynthesis.

Enhancement Agents

Production of secondary metabolites is a complex process, requiringcoordinated action of many different enzymes to produce and sequentiallymodify the precursors which are ultimately converted into the secondarymetabolites. At the same time, secondary metabolite production will belowered if other enzymes metabolize precursors of the desiredmetabolite, draining the precursor pools needed to build the secondarymetabolites.

Limitation of the amount of available precursor, due to low productionor subsequent diversion, or limitation in the conversion of a precursoror intermediate to a downstream intermediate, or limitation in theactivity of a given enzyme, will limit the production of secondarymetabolites. In any particular culture system, the rate at which asecondary metabolite is produced will be controlled by one of theselimitations, forming a bottleneck in the pathway by which theprecursor(s) are converted into the secondary metabolite. Relieving thelimitation which causes the bottleneck will increase the rate ofsecondary metabolite production in that culture system up to the pointat which another step in the pathway becomes limiting. The particularstep which limits the overall rate of production will vary betweendifferent cultures, as will the action which relieves the limitation.

Taxanes are secondary metabolites which are produced through a series ofmany enzymatic steps, and the present inventors have determined severalclasses of enhancement agents which relieve one or more of the ratelimiting steps in taxane biosynthesis. Addition of one of theseenhancement agents to a culture of taxane-producing cells will enhancethe rate of taxane production. Furthermore, the inventors havedetermined that use of the enhancement agents discussed herein will haveat least some enhancing effect in most taxane-producing cultures,suggesting that the overall production rate is determined not by asingle rate-limiting step, but by a complex interaction among amultiplicity of limiting factors. Relief of any one of the limitingfactors will enhance taxane production, although the magnitude of theenhancement will depend on particular culture conditions which determinethe relative limiting effects of other steps in taxane biosynthesis,once a particular limitation has been relieved. Culture conditions whichaffect the interaction between various limiting factors include thegenetic make up of the cells, the composition of the culture medium andthe gaseous environment, temperature, illumination and process protocol,and the enhancement agent(s) added to a particular culture will usuallybe selected in view of the limiting factors in that culture, which maybe determined empirically by comparing the effects of individualenhancement agents as set forth herein. Furthermore, it has beendiscovered that further enhancement of taxane production will beachieved if more than one enhancement agent is present in the culture.

Representative enhancement agents within the contemplation of thisinvention are exemplified in Table 1. The enhancement agents of thisinvention will be discussed under several general classes. These classesare: anti-browning agents, anti-senescence agents, anti-ethylene agents,plant growth regulators, such as auxin-related growth regulators,precursors, inhibitors, elicitors, stimulants and jasmonate-relatedcompounds.

One class of enhancement agents contemplated by this invention areanti-browning agents. As used herein, the term “anti-browning agents”refer to components that are added to the nutrient medium to prevent theformation of pigments during cell cultivation. These pigments includephenolics and related compounds that are generally observed to have adeleterious effect on cell growth, viability, and product formation. Atypical anti-browning agent used in the nutrient media according to thisinvention is ascorbic acid. Anti-browning agents may be typicallyincorporated in the medium at a concentration range of 10 ppb to 1000ppm.

Another class of enhancement agents is anti-senescence agents. Ananti-senescence agent is a compound of biological or non-biologicalorigin that protects cells from senescence. Such agents could act by,for example, blocking the production of compounds that promotesenescence, blocking the action of senescence-promoting factors,providing radical-scavenging or anti-oxidant activities, protecting theintegrity of cellular membranes and organelles, or by other mechanisms.Such agents include antagonists of ethylene action; polyamines and theirmetabolites, such as spermine, spermidine, diaminopropane, and the like;anti-browning agents, inhibitors of phenolics production, and radicalscavengers, such as reduced glutathione, propyl gallate, and sulfhydrylcompounds such as β-mercaptoethanolamine.

Anti-ethylene agents are defined as substances that interfere withethylene production or ethylene action. Anti-ethylene agents thatinterfere with ethylene Metabolism may be further classified asethylene-biosynthesis antagonists, and ethylene-action antagonists.Ethylene-biosynthesis antagonists are compounds that interfere with thebiosynthetic pathway to ethylene; examples of enzymes along thisbiosynthetic pathway that are inhibited include ACC synthase, ACCoxidase, and ethylene oxidase. Examples of ethylene biosynthesisantagonists include α-aminoisobutyric acid, acetylsalicylic acid,methoxyvinylglycine, aminooxyacetic acid and the like.

Examples of ethylene action antagonists include silver containingcompounds, silver complexes, or silver ions, carbon dioxide,1-methylcyclopropene, 2,5-norbornadiene, trans-cyclooctene, cis-butene,diazo-cyclopentadiene and the like. Suitable silver salts include silvernitrate, silver thiosulfate, silver phosphate, silver benzoate, silversulfate, silver salt of toluenesulfonic acid, silver chloride, silveroxide, silver acetate, silver pentafluoropropionate, silver cyanate,silver salt of lactic acid, silver hexafluorophosphate, silver nitrite,and the trisilver salt of citric acid. Illustrative examples of theenhancement of taxane biosynthesis by a variety of silver salts areshown in Example 10.

Anti-ethylene agents may be incorporated into the medium at levels of 10ppb to 1000 ppm. When silver is incorporated in the medium, it will beadded at a concentration of less than 900 μM, preferably less than 500μM, and more preferably less than 200 μM. When silver is incorporated inthe medium, it will be added at a concentration of at least 10 nM,preferably 100 nM, more preferably 1 μM, and typically at 10 μM.

Enhancement agents contemplated in this invention include plant growthregulators, particularly auxin-related growth regulators, which willinclude auxins, compounds with auxin-like activity, and auxinantagonists. Auxin-related growth regulators will typically beincorporated in the medium at concentrations of between 10⁻¹⁰ M and 10⁻³M, preferably between 10⁻⁸ and 10⁻¹⁰ M. Most preferred examples ofauxin-related growth regulators include 1-Naphthaleneacetic acid,2-Naphthaleneacetic acid, 1-Naphthaleneacetamide/Naphthylacetamide,N-(1-Naphthyl)phthalamic acid, 1-Naphthoxyacetic acid, 2-Naphthoxyaceticacid, beta-Naphthoxyacetic acid, 1-Naphthoxyacetamide,3-Chlorophenoxyacetic acid, 4-Chlorophenoxyacetic acid,3-Iodophenoxyacetic acid, Indoleacetamide, Indoleacetic acid,Indoylacetate, Indoleacetyl leucine, Gamma-(3-Indole)butyric acid,4-Amino-3,5,6-trichloropicolinic acid, 4-Amino-3,5,6-trichloropicolinicacid methyl ester, 3,6-Dichloro-o-anisic acid,3,7-Dichloro-8-quinolinecarboxylic acid, Phenylacetic acid,2-Iodophenylacetic acid, 3-Iodophenylacetic acid, 2-Methoxyphenylaceticacid, Chlorpropham, 4-chloroindole-3-acetic acid,5-Chloroindole-3-acetic acid, 5-Bromo-4-chloro-3-indoyl butyrate,Indoleacetyl phenylalanine, Indoleacetyl glycine, Indoleacetyl alanine,4-chloroindole, p-chlorophenoxyisobutyric acid, 1-pyrenoxylbenzoic acid,Lysophosphatidic acid, 1-naphthyl-N-methylcarbamate, andEthyl-5-chloro-1H-Indazole-3-ylacetate-3-Indolebutanoic acid. Otherpreferred examples of auxin-related growth regulators includeNaphthalene-2,6-dicarboxylic acid, Naphthalene-1,4,5,8-tetracarboxylicacid dianhydride, Naphathalene-2-sulfonamide,4-Amino-3,6-disulfo-1,8-naphthalic anhydride, 3,5-dimethylphenoxyaceticacid, 1,8-Naphthalimide, 2,4-Dichlorophenoxyacetic acid,2,3-Dichlorophenoxyacetic acid, 2,3,5-Trichlorophenoxyacetic acid,2-Methyl-4-chlorophenoxyacetic acid, Nitrophenoxyacetic acids,DL-alpha-(2,4-Dichlorophenoxy)propionic acid,D-alpha-(2,4-Dichlorophenoxy)propionic acid, 4-Bromophenoxyacetic acid,4-Fluorophenoxyacetic acid, 2-Hydroxyphenoxyacetic acid, 5-Chloroindole,6-Chloro-3-indoylacetate, 5-Fluoroindole, 5-Chloroindole-2-carboxylicacid, 3-Chloroindole-2-carboxylic acid, Indole-3-pyruvic acid,5-Bromo-4-chloro-3-indoylbutyrate, 6-Chloro-3-indoylbutyrate,Quinoline-2-thioglycolic acid, Aminophenylacetic acids,3-Nitrophenylacetic acid, 3-Chloro-4-hydroxybenzoic acid, Chlorflurenol,6-Chloro-3-indoyl acetate,N-(6-aminohexyl)-5-chloro-1-Naphthalenesulfonamide hydrochloride,2-chloro-3(2,3-dichlorophenyl)propionitrile, o-chlorophenoxyacetic acid,6,7-dimethoxy-1,2-benzisoxazole-3-acetic acid,3-oxo-1,2-benzisothiazoline-2-ylacetic acid, Mastoparan,2,3,5-Triidobenzoic acid, 2-(3-chlorophenoxy)propanoic acid, andMecoprop. Other examples of suitable auxin-related growth regulatorsinclude Naphthoic acid hydrazide, 2,4-Dibromophenoxyacetic acid,3-Trifluoromethylphenoxyacetic acid, Oxindole, Indole-2-carboxylic acid,Indole-3-lactic acid, Beta-(3-Indole)propionic acid, 2-Bromophenylaceticacid, 3-Bromophenylacetic acid, 2-Chlorophenylacetic acid,3-Chlorophenylacetic acid, 2-Methylphenylacetic acid,3-Methylphenylacetic acid, 3-Trifluoromethylphenylacetic acid,3-Methylthiophenylacetic acid, Phenylpropionic acid,4-chloro-2-methylphenylthioacetic acid, 2-Chlorobenzoic acid,3-Chlorobenzoic acid, 2,3-Dichlorobenzoic acid, 3,4-Dichlorobenzoicacid, 2,3,5-Trichlorobenzoic acid, 2,4,6-Trichlorobenzoic acid,2-Benzothiazoleoxyacetic acid,2-Chloro-3-(2,3-dichlorophenyl)propionitrile, 2,4-Diamino-s-triazine,Naphthalic anhydride, Dikegulac, chlorflurecolmethyl ester,2-(p-chlorophenoxy)-2-methylpropionic acid,2-chloro-9-hydroxyfluorene-9-carboxylic acid,2,4,6-trichlorophenoxyacetic acid, 2-(p-chlorophenoxy)-2-methylpropionic acid, Ethyl 4-(chloro-o-tolyloxy)butyrate,[N-(1,3-dimethyl-1H-Pyrazol-5-yl)-2-(3,5,6-Trichloro-2-pyridinyl)oxy]acetamide,4-Chloro-2-oxobenzothiazolin-3-yl-acetic acid,2-(2,4-Dichlorophenoxy)propanoic acid, 2-(2,4,5-Trichlorophenoxy)propanoic acid, 4-Fluorophenylacetic acid, 3-Hydroxyphenylacetic acid,Orthonil, 3,4,5-Trimethoxycinnamic acid,2(3,4-dichlorophenoxy)triethylamine, Indole-3-propionic acid, SodiumIoxynil, 2-Benzothiazoleacetic acid, and(3-phenyl-1,2,4-thiadiazol-5-yl)thioacetic acid.

Other classes of plant growth regulators may also be incorporated intothe nutrient medium as enhancement agents. These includecytokinin-related growth regulators such as N⁶-benzyladenine,6-[γ,γ-dimethylallylamino]purine, kinetin, zeatin,N-phenyl-N′-1,2,3-thidiazol-5-ylurea (thidiazuron) and relatedphenylurea derivatives and the like, gibberrellins such as GA₃, GA₄,GA₇, and GA derivatives, abscisic acid and its derivatives,brassinosteroids, and ethylene-related growth regulators. Such growthregulators may be incorporated in the medium at concentrations between10⁻¹⁰ M and 10⁻³M, preferable between 10⁻⁸M and 10⁻⁵M.

Another class of enhancement agents are precursors or biosyntheticprecursors. As used herein, the term precursors are used to describecompounds added to the nutrient medium that are metabolized andincorporated by the cells into taxol and taxanes. Suitable precursorsinclude precursors of isoprenoid compounds such as acetate, pyruvate andthe like; α-phenylalanine, β-phenylalanine (3-amino-3-phenylpropionicacid), phenylisoserine, N-benzoylphenylisoserine, benzoic acid, shikimicacid, glutamine, cinnamic acid, and the like. Derivatives of theaforementioned molecules are also suitable as precursors.

Another class of enhancement agents are inhibitors. Inhibitors arecompounds which inhibit enzymatic or other cellular activities As usedherein, the term “metabolic inhibitors” are used to describe compoundsadded to the nutrient medium that interfere with specific biosyntheticpathways. For example, a metabolic inhibitor may be used to enhancetaxol, baccatin III, or other taxane biosynthesis by blocking adifferent pathway that competes for an early biosynthetic precursor.Particularly effective enhancement agents of this class includeinhibitors of phenylpropanoid metabolism, which are compounds capable ofinhibiting the synthesis or metabolism of cinnamic acid or itsderivatives. These compounds include preferably p-Coumaric acid,4-Fluoro-DL-tyrosine, 4-Methoxybenzoic acid, 3-dimethylaminobenzoicacid, 4-methoxycinnanic acid, 4-nitrocinnamic acid ethyl ester,4-Nitrocinnamaldehyde, Mercaptoethanol, 4-hydroxycoumarin,Cinnamylfluorene, 2-cyano-4-hydroxycinnamic acid, Cinnamylidenemalonicacid, 4-dimethylaminocinnamic acid, N-cinnamylpiperazine,N-Trans-cinnamoylimidazole, 2-Aminoindan-2-Phosphonic acid,Benzylhydroxylamine, Procaine, Monensin, N-(4-Hydroxyphenyl)glycine,3-(4-hydroxyphenyl)propionic acid, 3-(2-hydroxyphenyl)propionic acid,more preferably D-Phenylalanine, N-(2-mercaptopropionyl)glycine and itsacetic acid salt complex, DL-Metafluorophenylalanine,p-Fluoro-DL-phenylalanine, Dithiothreitol, 4-Fluorocinnamic acid,Trans-3,4-Difluorocinnamic acid, 3,4-Difluoro-D-Phenylalanine,diethyldithiocarbamic acid, 4-Fluoro-(1-amino-2-phenylethyl)phosphonicacid, 3,4-methylenedioxybenzoic acid, and most preferably3,4-methylenedioxy-6-nitrocinnamic acid, 3,4-methylenedioxycinnamicacid, 3-[3,4-methylenedioxyphenyl]propionic acid,3,4-methylenedioxyphenylacetic acid, 4-Fluoro-L-Phenylalanine,4-Hydroxyphenylpyruvic acid, 4-Fluoro-DL-Tyrosine, Trans3,4-Dimethoxycinnamic acid, phenylpropiolic acid,L-2-Hydroxy-3-Phenylpropionic acid, 2-hydroxy-4,6-dimethoxybenzoic acid,SKF-525A (2-(diethylamino) ethyl ester of α-phenyl-α-propylbenzeneaceticacid), vinylimidazole, ammonium oxalate, sinapic acid, and1-aminobenzotriazole and related analogs. When incorporated into themedium, the inhibitors will be added at a concentration between 10 ppband 1000 ppm, preferably at a concentration between 100 ppb and 100 ppm,and more preferably at a concentration of 1 ppm to 50 ppm.

In order to improve the yield of taxol, baccatin III, and other relatedtaxanes in cell cultures, the inventors have undertaken a number ofapproaches. One of the approaches that has been used to enhanceproductivity is the use of so-called elicitors. As used herein, the termelicitors is used for compounds of biological and non-biological originthat cause an increase in secondary metabolite production when appliedto plants or plant-cell cultures (Eilert 1987, “Cell Culture and SomaticGenetics of Plants,” Vol. 4, F. Constabel and I. K. Vasil (eds.),Academic Press, New York, pp. 153-196; Ebel, 1984, Bioregulators:Chemistry and Uses. 257-271; and Darvill et al., 1984, Ann. Rev. PlantPhysiol., 35, 243-275). Many different compounds can act as elicitors,depending upon their nature of origin and their mode of action with cellmetabolism. In these studies, the inventors have used two major kinds ofelicitors: 1) Biotic elicitors which usually comprise cell wall extractsor filtrates from a selected group of fungi, bacteria and yeasts, andalso their purified fractions. 2) Abiotic elicitors which have includedchemical stress agents as well as some compounds of biological origin(see elicitors listed in Table 1). In addition, salts and complexescontaining heavy metal ions may also be considered as effective abioticelicitors; these include examples such as cobalt, nickel, lanthanum,selenium, vanadium, lead, cadmium, chromium, aluminium, iodine, barium,bismuth, lithium, rubidium, strontium, and gold. It should be noted thatcertain compounds that mediate elicitation, for example, thejasmonate-related compounds described below, may also be considered aselicitors.

Christen et al. (1991) report the use of fungal elicitors and selectedcompounds for production of taxol by suspensions of Taxus brevifolia;however, the increases in the level of taxol accumulation due toelicitor treatments have not been specified.

In general, both kinds of elicitors were effective, although the extentto which elicitation (taxane accumulation in cell cultures as well astheir secretion into the medium) occurred differed from elicitor toelicitor and from species to species. The highest production increasewas attained with chitosan glutamate, lichenan, ferulic acid and benzoicacid. Chitosan and lichenan are complex polysaccharides derived frommicrobial cell walls. Chitosan when used alone is insoluble in medium,and is toxic and causes permanent cell damage. Chitosan glutamate, onthe other hand, is readily soluble in medium and does not affect cellviability. Ferulic and benzoic acids are synthesized chemicals ofbiological origin, and are generally used as anti-oxidants in biologicalsystems.

Elicitors and metabolic stress agents may be utilized according to thisinvention to maximize taxol, baccatin III, and total taxane productionand secretion in tissue culture by assessing elicitor specificity andconcentration, timing, and duration, as a function of culture age andmedia composition.

Another class of enhancement agents contemplated in this invention arestimulants. As used herein the term stimulant is used to describecompounds added to the nutrient medium that stimulate or activatespecific biosynthetic pathways, for example those leading tobiosynthesis.

Jasmonate-related compounds are a class of compounds that mediate theelicitation reaction, thereby stimulating secondary metabolitebiosynthesis. Jasmonate-related compounds include jasmonic acid and itsalkyl esters, such as methyl jasmonate, ethyl jasmonate, propyljasmonate, butyl jasmonate, pentyl jasmonate, hexyl jasmonate;dihydrojasmonic acid and its alkyl esters, such as methyldihydrojasmonate, ethyl dihydrojasmonate, n-propyl dihydrojasmonate,butyl dihydrojasmonate, pentyl dihydrojasmonate, hexyl dihydrojasmonate;epimethyl jasmonate, fluoromethyl jismonate, cis-jasmone, isojasmone,tetrahydrojasmone, 12-oxophytodienoic acid, dihydrojasmone, jasmonylacetate, apritone, amylcyclopentenone, hexylcyclopentenone,hexylcyclopentanone, and related derivatives and analogs.Jasmonate-related compounds are incorporated into the medium atconcentrations of 10⁻⁹ M to 10⁻³ M and preferably at concentrations of10⁻⁶ to 5×10⁻⁴ M, and more preferably at concentrations of 10⁻⁵ M to2×10⁻⁴ M. It should be noted that more than one jasmonate-relatedcompound may be incorporated into the nutrient medium. It will berecognized by the skilled artisan that the concentration of enhancementagents such as jasmonate-related compounds, auxin-related growthregulators, precursors, and other nutrients will change as thesecompounds are metabolized in the culture. Unless otherwise indicated,the concentrations recited herein refer to the initial concentration inthe nutrient medium.

Combining enhancement agents from at least two of the following classesof enhancement agents has been shown to enhance taxane production byTaxus cells beyond the maximum enhancement observed for any one of theagents when used alone. These classes of enhancement agents areelicitors, jasmonate-related compounds, inhibitors of ethylene action,inhibitors of phenylpropanoid metabolism, antisenescence agents,precursors and auxin-related growth regulators. Therefore, in apreferred mode, this invention provides methods for enhancing productionof one or more taxanes by culturing cells of a Taxus species in thepresence of enhancement agents selected from at least two of these agentgroups.

Preferred methods for taxane production use the prototype inhibitor ofethylene action, silver, in combination with at least one otherenhancement agent, and in particularly preferred methods the other agentis methyl jasmonate, or an inhibitor of phenylpropanoid metabolism, suchas 3,4-methylenedioxynitrocinnamic acid.

When used in combination with each other, jasmonate-related compoundsand ethylene-action inhibitors may be incorporated into the nutrientmedium in certain proportions to each other. For example, when methyljasmonate and silver thiosulfate are used in combination, the molarratios of methyl jasmonate to the silver ion may be in the range between0.0001 to 9.5, preferably in the range between 0.001 to 8, morepreferably in the range between 0.1 to 7, and most preferably in therange between 1 to 5.

When used in combination with each other, auxin-related growthregulators and ethylene-action inhibitors may be incorporated into thenutrient medium in certain proportions to each other. For example, whenan auxin-related growth regulator and silver thiosulfate are used incombination, the molar ratios of auxin-related growth regulator tosilver ion may be in the range between 0.011 to 1000, preferably in therange between 0.015 to 100, and more preferably in the range between0.02 to 50, and most preferably between 0.05 to 30.

Generally, when culturing of Taxus cells for the production of taxanes,one or more auxin-related growth regulator will be added to the culturemedium. Presence of auxin-related growth regulator(s) will promote cellgrowth, but more significantly will enhance production of taxanes by theculture. Further enhancement can be obtained by adding at least oneother enhancement agent contemporaneously with the auxin-related growthfactor.

In a preferred mode of this invention, one or more enhancement agentsare added to the culture in an amount sufficient to enhance theproduction of one or more taxanes by at least 3-fold, preferably by atleast 5-fold, more preferably by at least 10-fold, and even morepreferably by at least 30-fold relative to the level of production inthe absence of the enhancer(s). In another preferred mode of thisinvention, one or more enhancement agents are added to the culture in anamount sufficient to enhance the volumetric productivity of taxol atleast to 10 mg/L/day, more preferably to at least 15 mg/L/day, and evenmore preferably to at least 22 mg/L/day. In another preferred mode ofthis invention, one or more enhancement agents are added to the culturein an amount sufficient to enhance the whole broth titer of taxol to atleast 150 mg/L, more preferably to at least 200 mg/L, and even morepreferably to at least 350 mg/L. In another preferred mode of thisinvention, one or more enhancement agents are added to the culture in anamount sufficient to enhance the volumetric productivity of baccatin IIIto at least 15 mg/L/day, more preferably to at least 20 mg/L/day, andeven more preferably to at least 25 mg/L/day. In another preferred modeof this invention, one or more enhancement agents are added to theculture in an amount sufficient to enhance the whole broth titer ofbaccatin III to at least 100 mg/L, more preferably to at least 150 mg/L,and even more preferably to at least 250 mg/L. In another preferred modeof this invention, one or more enhancement agents are added to theculture in an amount sufficient to enhance the volumetric productivityof taxanes to at least 15 mg/L/day, more preferably to at least 25mg/L/day, and even more preferably to at least 40 mg/L/day. In anotherpreferred mode of this invention, one or more enhancement agents areadded to the culture in an amount sufficient to enhance the whole brothtiter of taxanes to at least 200 mg/L, more preferably to at least 300mg/L, and even more preferably to at least 400 mg/L.

Many of the compounds described as enhancement agents above have beenused in other plant systems. Formulation, administration, andappropriate physiological concentration levels in these non-Taxussystems will provide guidance for the skilled artisan to apply theseagents in accordance with this invention.

Cellular Material

Suitable cells for culture in the method of this invention may be fromany species of Taxus. Preferably, the cells will be from a cell linethat inherently produces taxanes in relatively high yield. Typically,such cells have the ability to produce high levels of one or moretaxanes under standard conditions or exhibit high average volumetricproductivities of taxanes under standard conditions. Suitable cell linesmay be identified by culturing cells of the cell line under standardtaxane production conditions and observing the level of one or moretaxanes produced in the culture or determining the average volumetricproductivity for one or more taxanes by the in the culture the followingprocedures.

Cells for use in the production culture testing procedure are grown in asuitable medium adapted for the particular cell line. Followingcompletion of log phase growth, an aliquot of cells are cultured fortest production of taxanes. Production culture is generally performed inliquid medium, although callus culture on solid medium may be used. Inproduction culture, the cells are cultivated in medium N from Table 2,in medium N from Table 2 except for replacement of sucrose by 7% (w/v)maltose, or in a nutrient medium optimized for growth and maintenance ofthe particular cell line. In the production culture, the cell densityshould be in the range of 15-20 percent (w/v) on a fresh weight basis.Cells are cultured for 10-20 days at 25 C+/−2 C under dark conditions.Liquid cultures should be appropriately agitated and aerated, forexample on a rotary shaker at 120-180 rpm.

Production cultures for evaluating cell line characteristics willinclude suitable enhancement agents. Generally, six alternativeenhancement cocktails (combinations of up to five enhancement agents)are tested for each cell line. The combinations are shown in Table Abelow.

At the end of the culture, titer of individual taxanes in the culturemay be measured by ELISA assay performed as described herein, or theprofile of taxanes produced in the culture may be determined by HPLCanalysis as described in Example 5. Preferred cell lines will produceone or more taxanes above the minimum target taxane levels in one ormore of the enhancement cocktails. Preferred cell lines will exceed thetarget levels for both titre and productivity for at least oneenhancement cocktail, and more preferably for two or more enhancementcocktails. Minimum target taxane titer at the end of production culturefor suitable cell lines will be at least 100 mg/L taxanes.Alternatively, the minimum average volumetric productivity target overthe course of the production culture will be 10 mg/L/day taxanes. Morepreferred cell lines will achieve minimum taxane titer at the end ofproduction culture of at least 100 mg/L taxol or 200 mg/L baccatin III,or average volumetric productivity over the course of the productionculture of 10 mg/L/day taxol or 15 mg/L/day baccatin III.

TABLE A Enhancement Cocktails Combinations of Enhancement Agents: 1. 20μM Naa + 30 μM Mdna 2. 20 μM Naa + 30 μM Mdna + 50 μM Slts 3. 20 μMNaa + 30 μM Mdna + 89 μM Mjs 4. 20 μM Naa + 30 μM Mdna + 89 μM Mjs + 50μM Slts 5. 20 μM Naa + 30 μM Mdna + 89 μM Mjs + 50 μM Slts + 5 mM Gln 6.20 μM Naa + 89 μM Mjs + 50 μM Slts Gln = glutamine Naa =1-naphthaleneacetic acid Mdna = 3,4-methylenedioxy-6-nitrocinnamic acidMjs = methyl jasmonate Slts = silver thiosulfate

Suitable production media for the various species are listed in Table 5,although others may be used. For example, Media B, C and N from Table 2are particularly suitable production media for Taxus chinensis. Mediapreferably contain the ingredients listed in Table 2. These mediapreferably contain major and minor inorganic salts, organics and growthhormones or growth regulators, in the amounts generally with thepreferred ranges starting with the 1/10th to three times theconcentration of each medium ingredient indicated in Table 2. Wheremedium B or N is used, the growth regulators are typically incorporatedinto the medium in an amount between 0.1 ppm to 20 ppm, and preferablybetween 1 ppm to 10 ppm. When Medium C or N is used, the growthregulators are incorporated preferably at levels ranging from 0.1 ppm to5 ppm.

It will be understood by the skilled artisan that within thecontemplation of this invention modifications may be made in the mediadescribed herein, such as substitution of other conventionalcompositions (such as organics, vitamins, amino acids, precursors,activators and inhibitors), addition or deletion of various components,including growth regulators, or alteration of proportions, so as toproduce growth and taxane production equal to or better than thatobserved with the media in Table 2.

Modes of Process Operation

The operating mode for a plant cell culture process refers to the waythat nutrients, cells and products are added or removed with respect totime (Payne et al. 1991). When all the nutrients are supplied initially,and the culture contents comprising cells and product are harvested atthe end of the culture period, the operating mode is termed a “one-stagebatch process”. When a batch process is divided into two sequentialphases, a growth and a production phase, with the medium being exchangedin between the two phases, the operating mode is termed a “two-stagebatch process”. Within the contemplation of this invention, thetransition from the growth medium through production medium, may occurby an abrupt stepwise change, or progressively by a series of continuoussteps, or by progressive change. In one extreme the progressive changeis accomplished by progressive replacement of medium, of incrementallychanging composition. In another alternative, the progressive change isaccomplished by feeding one or more components of the production mediuminto the growth phase culture. This is one example of the fed-batchprocess.

In a “fed-batch” operation, particular medium components such asnutrients and/or one or more enhancement agents are supplied eitherperiodically or continuously during the course of a culture. It shouldbe noted that certain components may be incorporated into the nutrientmedium initially in the batch mode, then added in fed-batch mode, or maybe added to the nutrient medium exclusively in the fed-batch mode.

Using fed-batch operation, it has been found that cells can be sustainedin a productive state for a prolonged period, and in fact, thatproductivity of the cells could be enhanced. As illustrated in Examples15 and 17, and in Tables 16 and 18, adding certain nutrients andenhancement agents in a fed-batch manner gave significant improvementsin overall performance for taxanes generally, and for specific taxanessuch as taxol and bacctin III. Further, this mode of operation has beenfound to be compatible with a variety of different cell lines under avariety of different media conditions.

Fed-batch addition of components is particularly advantageous when theconcentration of the particular component has to be maintained at a lowlevel in the culture, for example, to circumvent the effects ofsubstrate inhibition. Similarly, fed-batch addition is advantageous whencells react negatively to a component when it is either added initiallyto the nutrient medium or if stoichiometrically-meaningful quantities ofa component cannot be added due to solubility or toxicity limitations.Further, continuous or continual (periodic) fed-batch addition of a feedsolution containing a component is particularly preferred when cellsreact negatively to the component when it is added in a more rapidmanner such as pulse addition. Particular components to which cellsrespond favorable when added in a fed-batch mode include taxaneprecursors such as alpha- and beta-phenylalanine; carbon sources such asmaltose, fructose and glucose; amino acids such as glutamine, glutamicacid, aspartic acid; macronutrients such as phosphate, calcium, andmagnesium; and enhancement agents such as auxin-related growthregulators and jasmonate-related compounds.

It will be apparent to the skilled artisan, that the composition of thefeed may be varied to obtain the desired results such as extension ofthe production phase to increase taxane yield or extension of the growthphase to achieve higher biomass density. Selection of suitableconditions to achieve optimum productivity and performance is easilywithin the skill of the ordinary artisan in view of the teachingsdescribed herein. Similarly variations of other operating parameters,such as the timing and duration of the addition and the rate of theaddition of the fed-batch components, to achieve the desired results,are within the reach of the skilled artisan in view of the teachingsdescribed herein.

Medium exchange as described herein refers to the removal of spentmedium from the culture followed by addition of fresh medium to theculture; the cells are largely retained in the culture during theoperation. In the method of this invention, medium exchange operation isan advantageous method to obtain and sustain high volumetricproductivities of taxane production, resulting in superior processperformance and overall production levels, compared to a batch process.The extracellular product resulting from such an operation may lenditself to more facile downstream recovery and purification than otherprocess modes.

As illustrated in Example 14 and Table 15, medium exchange is successfulin sustaining high productivities for taxanes generally, and forspecific taxanes such as taxol, baccatin III, and 10-deacetylbaccatinIII. In addition, this mode of operation resulted in the increase in thevolumetric productivity relative to batch operation for taxanesgenerally, and for specific taxanes such as taxol and baccatin III.Further, this mode of operation is compatible with a variety ofdifferent cell lines under a variety of different media conditions. Asfurther illustrated in Example 7.3, the removal of spent medium andreplenishment of fresh medium every 3 days contributed to significantenhancement of taxane and taxol production in growth conditions, as wellas to an increase in the amounts of extracellular product.

The stimulatory effects of medium exchange may have been due to removalof product in situ, which would prevent feedback inhibition and productdegradation. Such positive effects of in situ product removal onsecondary metabolite production and secretion in suspension cultureshave been documented by, among others, Robins and Rhodes (1986, Appl.Microbiol. Biotechnol., 24, 35-41) and Asada and Shuler (1989. Appl.Microbiol. Biotechnol., 30, 475-481). The periodic removal of spentmedium incorporates the above advantages, and additionally, may serve tode-repress secondary biosynthesis by removing other, non-taxane,inhibitory components (such as phenolic compounds) from the medium.

The replenishment of fresh medium to cells undergoing activebiosynthesis may also enhance production by providing essentialnutrients that have been depleted. For example, Miyasaka et al. (1986,Phytochemistry, 25, 637-640) were able to stimulate stationary phasecells of Salvia miltiorhiza to produce the diterpene metabolites,cryptotanshinone and ferruginol simply by adding sucrose to the medium.Presumably, biosynthesis had ceased due to carbon limitation in thestationary phase. The periodic-medium-exchange protocol used in thepresent work could have been beneficial as a result of any of the abovefactors. It is understood that the amount of medium exchanged, thefrequency of exchange, and the composition of the medium beingreplenished may be varied. The ability to stimulate biosynthesis andsecretion by medium exchange has important implications for the designand operation of an efficient commercial process in the continuous,semi-continuous or fed-batch mode.

When a substantial portion, but not all, of the contents of a batchculture is harvested, with addition of fresh medium for continued cellgrowth and production, the process resembles a “repeated draw and fill”operation, and is termed a “semi-continuous process”. When fresh mediumis continuously supplied, and effluent medium is continuously removed,the process is termed “continuous”. If cells are retained within thereactor, the process is termed a “perfusion mode” If cells arecontinuously removed with the effluent medium, the continuous process istermed a “chemostat”.

It is understood that these various modes of process operation arecompatible with the taxane-production system described herein.

EXAMPLES

The following examples are provided to further describe the materialsand methods which may be used in carrying out the invention. Theexamples are intended to be illustrative and are not intended to limitthe invention in any manner.

Example 1 Callus Initiation

Samples of Taxus plant material were collected from a number of wild andcultivated plants. Samples were processed upon arrival at the laboratoryor stored at 4° C. until they could be used.

The material was first washed in dilute soap solution, rinsed in water,and the surface sterilized in a CLOROX solution (1% hypochlorite, pH 7)for 10 minutes. Under sterile conditions the material was then rinsed 3times with sterile water. Needles were then cut in a 1%polyvinylpyrrolidone (PVP) solution with 100 mg/l ascorbic acid. Needleswere placed with the cut end in Medium E (see Table 2). Thirty to fortyexplants were cultured per plate of medium. Plates containing explantswere incubated at 25±1° C. in the dark. Plates were monitored daily forthe appearance of contaminating micro-organisms, and where they werepresent, uncontaminated needles were removed and placed in a fresh plateof Medium E. Substantial callus formation was observed and the calluswas separated from the explant at 20 days and placed on the variouscallus proliferation media listed in Table 3. For example, calli ofTaxus chinensis were transferred to Medium D (see Table 2). Thisinitiation procedure was very efficient, resulting in low contaminationrate and high frequency of callus induction of over 90% of explantsinitiated. The same procedure was successfully used to initiate culturesof Taxus brevifolia, Taxus canadensis, Taxus cuspidata, Taxus baccata,Taxus globosa, Taxus floridana, Taxus wallichiana, Taxus media, andTaxus chinensis.

Example 2 Callus Proliferation

Once calli were removed from the explant, they were cultivated at 25+1°C. in the dark. Healthy parts of the callus were transferred to freshmedium every 7 to 10 days, and this frequency of transfer was found tobe extremely important for prevention of browning and for prolongedcallus maintenance. The preferred growth and maintenance media for calliof various species are summarized in Table 3.

Example 3 Suspension Initiation

1 g fresh weight of callus material was aseptically inoculated into a125 ml Erlenmeyer flask containing 25 ml of liquid medium appropriate toeach species (see Table 3). For example, Medium D was used for Taxuschinensis. The flask was covered with a silicone foam cap (Bellco, N.J.)and placed on a gyratory shaker at 120 rpm at 24±1° C. in darkness.Suspension cultures were formed in approximately 3 to 10 days.Initially, medium was exchanged by suction filtering the flask contentsthrough a buchner funnel containing a miracloth filter (Calbiochem), andresuspending all the biomass in fresh medium. Upon cell growth, 1-2 g(fresh weight) of cells, and were generally transferred into a new 125ml flask containing 25 mL of fresh medium and were thereaftersubcultured weekly.

Example 4 Growth of Suspended Cells

The typical growth rates and cell densities achieved in suspensioncultures of representative species are listed in Table 4.

As a detailed example, the increase in biomass (fresh and dry weight)with time for Taxus chinensis line K-1 is shown in FIG. 1. The maximumgrowth rate was measured by taking the slope at points of most rapidbiomass increase on the growth curves. Cell cultures of Taxus chinensisgrew at a maximum doubling time of 2.5 days. This growth rate issignificantly higher than that reported previously for Taxus speciessuspension cultures. For example, Christen et al. (1991) reported a 5-to 10-fold increase in biomass after 3 to 4 weeks of culture, whichtranslates to an average doubling time for Taxus brevifolia suspensionsof 7 to 12 days.

The ability to cultivate cells at a high density is important inmaximizing the volumetric productivity of a cell culture process. Whilecultures of Taxus brevifolia reached a cell density of less than 1 g dryweight per liter (calculated from data presented in Christen et al.(1991)), suspensions of Taxus chinensis were able to reach densities ofup to 8 to 20 g dry weight per liter after 18 days of growth. Theviability of cells was determined by staining cells with a 0.05%solution of fluorescein diacetate in acetone (Widholm, 1972, StainTechnol., 47, 189-194), and by counting the number of green fluorescingcells upon excitation with blue light in an inverted fluorescencemicroscope (Olympus IMT-2, Japan). Cell viability was higher than 90%throughout the growth phase.

The ability to cultivate cells under rapid growth conditions to highcell densities while retaining high viability is an importantpre-requisite to the economic operation of a plant cell culture processfor producing taxol, baccatin III, and taxanes.

Example 5 Analysis of Taxol, Baccatin III and Other Taxanes 5.1. ELISAMethods

ELISA analysis (Hawaii Biotech #TA-01) was used for detection of taxolin cell culture extracts (see Grothaus, et al., 1995). This methodprovides high sensitivity (0.1 ng/mL), however, because a polyclonalantibody is used, cross-reactivity with other taxanes is observed.Preparative (analytical scale) HPLC with fraction collection showedcross-reactivity with 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol,cephalomannine, 10-deacetyl-7-epitaxol, 7 epitaxol, as well as otherunidentified taxanes. Despite such cross-reactivity this method wasfound to be extremely useful for detection of taxane production andallowed large numbers of cell lines to be screened quickly. Cellextracts showing significant production of taxanes were then analyzed indetail using the HPLC procedures outlined below.

A monoclonal ELISA analysis (Hawaii Biotech #TA-02) was also used fordetection of taxol in cell culture extracts. This method provides highsensitivity (0.1 ng/mL) and significantly less cross-reactivity.

5.2. Extraction of Taxol, Baccatin III, and Other Taxanes

Extraction of taxanes from supernatants were performed by severalmethods depending on the concentrations present. When sufficient amountsof taxanes (approx. 1-5 mg/L) are present in liquid media, samples wereprepared very rapidly and efficiently. Media (2 mL) were driedcompletely (in vacuo) and a measured amount of methanol (0.5-2.0 mL) wasadded. This mixture was agitated ultrasonically until completedissolution or dispersion of the sample was accomplished. Solids wereremoved by centrifugation prior to HPLC analysis. Quantitativerecoveries have been obtained at 1 mg/L levels with detection levelswell below 0.1 mg/L.

When concentration of taxanes in the culture supernatants were very low(less than 1 mg/L), the medium was extracted three times with an equalvolume of a mixture of methylene chloride and isopropyl alcohol (IPA)(9:1 by vol.). The organic layer was reduced to dryness andreconstituted in a measured volume of methanol (50-250 mL). Multipleextraction typically recovered 90-95% of the taxol, cephalomannine, andbaccatin III at 0.6 mg/L levels.

When taxane concentrations in the supernatant exceeded ˜5 mg/L a morerapid sample preparation was employed. One part (vol.) of supernatantwas mixed with 3 parts (vol.) of methanol containing 0.1% acetic acid.This mixture then was sonicated for 30 minutes, filtered, and analyzedby HPLC.

Samples of whole broth (culture supernatant containing cells) wereprepared using a method similar to that described in the precedingparagraph. One part (vol.) of whole broth was mixed with 3 parts (vol.)of methanol containing 0.1% acetic acid. This mixture then was sonicatedfor 30 minutes, allowed to stand for an additional 30 minutes, filteredand then analyzed by HPLC.

Cell materials were extracted by freezing freshly harvested cells (−5°C.), followed by vacuum drying, and methanol soxhleting for 50 cycles.The volume of methanol was reduced (−100 fold) by rotary evaporation andthe resulting sample was analyzed by HPLC. 70 to 80% of the taxanes weregenerally recovered with 10-15% measurable decomposition. It was laterfound that exhaustive drying of the sample prior to soxhlet resulted inless than 5% degradation of taxol

The extraction of solid media and callus was accomplished identically tothat of cells when taxane levels were low, however, methylenechloride/IPA vs. water partitioning of the final methanol extract wasalways performed. When taxane levels exceeded ˜5 mg/L the whole brothextraction method was employed to prepare samples of callus onsolidified medium.

5.3. High Performance Liquid Chromatography Methods

Analytical high performance liquid chromatography (HPLC) was performedon a high-carbon loaded diphenyl column (Supelco, 5 mM, 4.6 mm×25 cm)with an LDC Analytical binary gradient high pressure mixing systemconsisting of CM3500/CM3200 pumps, a CM4100 variable volume autosamplerand an SM5000 photo diode array detector interfaced to a personalcomputer. Column temperature was regulated at 35° C. with an Eldex CH150column oven. Quantitative HPLC analysis of taxanes was accomplishedusing a binary gradient elution scheme as follows:

Time % Eluant A % Eluant B Flow 0 75 25 1 mL/min 40 35 65 ″ 42 25 75 ″47 25 75 ″ 50 75 25 ″ Eluant A = 0.015 mM KH₂PO₄ brought to pH 3.5 withtrifluoroacetic acid Eluant B = acetonitrile

The chromatographic methods used resemble several published methods(Witherup et al. 1989, J. Liq. Chromatog., 12, 2117-2132) with theexceptions that a phosphate buffer containing trifluoroacetic acid hasbeen used and that a longer gradient is employed. These differencessignificantly improve the resolution of taxol and other taxanes from themixture. The relative retention times observed for taxanes are shownbelow. Taxol elutes between 31 and 33 minutes depending on the columnand hardware used.

Compound Relative Retention Time 10-deacetylbaccatin III 0.38 baccatinIII 0.56 7-xylosyl-10-deacetyltaxol 0.80 10-deacetyltaxol 0.87cephalomannine 0.94 10-deacetyl-7-epitaxol 0.98 taxol 1.00 7-epitaxol1.12

The retention times of taxol, cephalomannine and baccatin III weredetermined using authentic samples obtained from the National CancerInstitute. The retention times of the other taxanes listed above werecompared to analytical standards provided by Hauser Chemical (Boulder,Colo.). Identification of known taxanes was based on retention time andultraviolet spectral comparisons. Compounds that exhibited a UV spectrumsimilar to that of taxol and baccatin III, but that did not correlate tothe relative retention times of these taxanes were considered taxanes.Quantitation of taxol, cephalomannine and baccatin III was based onresponse factors determined from authentic materials. Quantitation of10-deacetylbaccatin III was performed using the response factordetermined for baccatin III. Where appropriate, quantitation of theremaining taxanes was based on the response factors measured for taxoland baccatin III. The term total taxanes represents the sum of thetaxanes that exhibited a characteristic UV similar to taxol and baccatinIII. Total taxanes identified in Taxus cultures include, among others,10-deacetylbaccatin III, 9-dihydrobaccatin III,7-epi-10-deacetylbaccatin III, baccatin III, 9-dihydro-13-acetylbaccatinIII, 7-xylosyl-10-deacetylcephalomannine, 7-xylosyl-10-deacetyltaxol,7-epibaccatin III, 10-deacetyltaxol, 7-xylosyltaxol, cephalomannine,7-epi-10-deacetyltaxol, taxol, 2-benzoyl-2-deacetyl-1-hydroxybaccatin I,taxol C, 7-epitaxol, and 2-benzoyl-2-deacetylbaccatin I.

Taxanes that did not exhibit the characteristic UV absorbance, but didexhibit characteristics taxane-mass-fragmentation characteristics uponmass spectrometry, were also observed in Taxus cell cultures. Examplesof such taxanes produced in Taxus cell cultures are, among others,Taxuyunnanine C, and its analogs and derivatives.

Each of the standards (10 uL) was typically injected (initially thenafter 3 or 4 samples) and areas for each of the three components wereintegrated from the 227 nm chromatogram. Response factors for each ofthe components was obtained by linear least-squares analysis of thedata. 10 uL of each sample was injected and the amount per injection wascalculated based on the standard data regression. These results wereconverted to amount per liter or percent dry weight. FIG. 4 illustratesa typical chromatogram of a supernatant sample.

5.4 Rapid High Performance Liquid Chromatography Methods

In addition to the above method, several rapid methods of HPLC analysiswere developed to allow greater sample throughput. Two of these methodsare described in detail below.

Method 1). Rapid high performance liquid chromatography (HPLC) wasperformed on a Phenomenex Curosil-G column (5 uM, 4.6 mm×25 cm with 4.6mm×3 cm guard) at ambient temperature using the hardware describedabove. Quantitative HPLC analysis of taxanes was accomplished using abinary gradient elution scheme as follows:

Time % Eluant A % Eluant B Flow 0 60 40 1.5 mL/min 10 25 75 ″ 11 25 75 ″Eluant A = 0.01 mM KH₂PO₄ brought to pH 3.5 with trifluoroacetic acidEluant B = acetonitrile

The relative retention times observed for taxanes are shown below. Taxolelutes at about 8 minutes depending on the column and hardware used.

Compound Relative Retention Time 10-deacetylbaccatin III 0.42 baccatinIII 0.61 taxol 1.00

Standards containing taxol, baccatin III and 10-deacetylbaccatin IIIwere prepared at 50 mg/L, 10 mg/L, and 1 mg/L levels. A standard wasinjected initially and then after every ninth sample and areas for eachof the three components were integrated from the 227 nm chromatogram.Response factors for each of the components was obtained by linearleast-squares analysis of the data. 10 μL of each sample was injectedand the amount per liter was calculated from the peak area based on thesample dilution and the standard data regression.

Method 2). Rapid high performance liquid chromatography (HPLC) was alsoperformed on a Phenomenex IB-SIL Phenyl column (3 uM, 4.6 mm×15 cm with4.6 mm×3 cm guard) at ambient temperature using the hardware describedabove. Quantitative HPLC analysis of taxanes was accomplished using abinary gradient elution scheme as follows:

Time % Eluant A % Eluant B Flow 0 65 35 1.0 mL/min 10 30 70 ″ 12 30 70 ″Eluant A = 0.015 mM KH₂PO₄ brought to pH 3.5 with trifluoroacetic acidEluant B = acetonitrile

The relative retention times observed for taxanes are shown below. Taxolelutes at about 9.5 minutes depending on the column and hardware used.

Compound Relative Retention Time 10-deacetylbaccatin III 0.41 baccatinIII 0.61 taxol 1.00

Quantitation was performed as described above.

Modifications of the above methods with respect to flow rate andgradient span and time were also found to perform suitablechromatography for plant cell culture analysis.

5.4. MS/MS Confirmation of Taxol

The identity of taxol in cell culture supernatant has been confirmedusing an MS/MS method (as shown in FIG. 6) which couples flow injectionwith ion spray atmospheric pressure chemical ionization. Details of theprocedures used for acquiring the data presented in FIG. 6 were asfollows: Mass Spectrometer: Sciex API 3 triple quadrupole with anatmospheric pressure ionization source. Nitrogen was used as the curtaingas and argon was used as the collision gas for the CID spectra.Interface: Ion Spray interface producing ions by Ion EvaporationIonization (Electrospray). Zero air was used as the nebulizer gas. LCPump: ABI 140B dual syringe pump operating at 5 μL/minute. Solvents:50/50 acetonitrile/H2O 2 mM NH4OAc+0.1% formic acid. Injection Volume: 5μL, all spectra taken by flow injection analysis. This method providedunequivocal confirmation for the presence of taxol in cell culturesamples, and also provided quantitation with excellent agreement to HPLCresults.

Example 6 Taxol Production by Various Species

The taxol produced by cell cultures of various Taxus species issummarized in Table 5. Callus was cultivated for 20 days in the dark onthe indicated solidified medium for each species. The cells and mediumwere dried and methanol-extracted together, and assayed by either ELISAor HPLC as indicated.

Example 7 7.1. Production in Growth Medium

The production of taxol and taxanes commenced within the first 2 days oftransfer into growth of Taxus chinensis cell line K-1 into Medium A. Themaximum taxol observed was on day 15, at 8.81 μg/flask, whichcorresponds to 0.44 mg/liter taxol. Of this, 46.1% was present in theextracellular medium. On day 15, the total taxane concentration was72.87 μg/flask, or 3.6 mg/liter, of which 58.6% was present in theextracellular medium. The viability of cells was always greater than 90%as measured by fluorescence staining (Example 4), suggesting that thepresence of extracellular taxol and taxanes was due to secretion ratherthan due to cell lysis.

The production levels of taxol, baccatin III, and related taxanes havebeen characterized for numerous different cell lines under a number ofdifferent growth conditions (elaborated in Table 2 and in otherexamples) in which taxane biosynthesis is not enhanced. These collectivedata indicate that when cultures are cultivated under conditionsoptimized for growth, but not for taxane biosynthesis, taxol productionlevels are typically less than or equal to 0.5 mg/L, and always lessthan or equal to 2 mg/L; the taxol volumetric productivities typicallyrange from 0.03 mg/L/day to 0.07 mg/L/day, and are always less than 0.3mg/L/day. Similarly, baccatin III production levels are typically lessthan or equal to 0.5 mg/L, and always less than or equal to 1 mg/L; thebaccatin III volumetric productivities are typically less than or equalto 0.03 mg/L/day, and always less than 0.15 mg/L/day. Similarly,total-taxane titers are typically less than 5 mg/L, and are always lessthan or equal to 20 mg/L; the total taxane volumetric productivities aretypically less than 1 mg/L/day, and always less than 3 mg/L/day.

7.3. Medium Exchange for Productivity Enhancement

Significant improvements in taxol and total taxane productivity wereobtained by aseptically suctioning off growth Medium A on day 9,replacing with fresh medium and repeating the procedure on day 12. Theexperiment was terminated on day 15, and the results are shown in FIG.2. The important increases in productivity due to medium exchange aresummarized in Table 6. The total amounts of taxol and taxanes producedwere ca. 4.6-fold higher with medium exchange compared to controlswithout treatment. Importantly, ca. 4.9-fold higher taxol, and ca.5.9-fold higher total taxanes were recovered in the extracellular mediumcompared to controls without medium exchange treatment.

The ability to markedly enhance taxol and total taxane productivities,and moreover, to cause extracellular product accumulation is importantfor operation of an efficient, continuous process with biomass reuse andsimplified downstream purification.

7.3. Effect of Light on Taxane Production in Growth Medium

Light is known to play an important role not only in photosynthesis, butalso in various aspects of secondary metabolism in plant cell cultures(Seibert and Kadkade 1980). Whereas the experiments described inExamples 4, 7.1, and 7.2 were conducted in darkness, the response ofTaxus chinensis cultures to light is described here.

One gram fresh weight of 7-day old cells of Taxus chinensis line K-1were inoculated in 25 ml of growth Medium A (see Table 2) in 125 mlErlenmeyer flasks and incubated at 24±1° C. on a gyratory shaker at 120rpm. Duplicate flasks were placed in the dark and under a StandardGroLux lamp at a distance of 3 feet. Spectral characteristics of thelamp are shown in FIG. 3. Results are shown in Table 7.

Exposure of cultures to light did not affect total taxane levels or theextent of extracellular accumulation. However, taxane profiles weresignificantly altered in the two treatments. For example, cellscultivated in the light produced 2.8 fold higher taxol than did cells inthe dark. The proportion of extracellular taxol was also significantlyhigher than in the dark treatment (76% vs 56%). The use of lighttreatment, especially of specific spectral quality, might be useful in acell culture process for taxol production.

Example 8 Elicitors

The term elicitors is used for compounds of biological (or biotic) andnon-biological (or abiotic) origin that cause an increase in secondarymetabolism when added to plant cell cultures.

While a number of elicitors have been found useful, a representativeillustrative example is described here in detail, namely, the use ofchitosan glutamate. While chitosan has been previously tried as anelicitor in some plant cell culture systems, the accompanying toxicreactions such as browning and loss of viability have made its useimpractical (Beaumont and Knorr 1987, Biotechnol. Lett. 9, 377-382).Indeed such toxic side reactions are a common drawback of many elicitorsreported in the literature. The use of chemically modified chitosanssuch as chitosan glutamate to specifically induce taxol and taxanebiosynthesis while circumventing toxic side-effects is a novel approach.

Suspensions of Taxus chinensis line K-1 grown in Medium D for 7 to 8days were suction filtered aseptically using a sterile Buchner funnelfitted with a miracloth (Calbiochem) filter. 2 g fresh weight cells wereaseptically transferred to 25 ml of medium C (see Table 2) in a 125-mLErlenmeyer flask. A solution of 0.05% chitosan glutamate was preparedfreshly and filter-sterilized through a 0.22 micron cartridge filter.825 μL of this solution was added to the flask at the start of theexperiment, corresponding to a level of 165 mg elicitor per gram dryweight cells. The flasks were incubated at 24±1° C. on a gyratory shakerat 110 rpm in the dark. The flasks were destructively sampled on day 15,and observations on growth, color of the cells and medium and cellviability were recorded. Samples were analyzed for taxanes as describedin Example 5. The results of this experiment are shown in Table 8.

Elicitor treatment resulted in a modest improvement in the per-celltotal taxane production (0.53% vs. 0.42% dry weight taxanes) overnon-treated controls. The non-toxic nature of the elicitor is evidentfrom the high viabilities (75-80%) observed in both treatments. In fact,an increased dry weight in elicitor treatment compared to controls hasbeen reproducibly observed (14.2 g/l vs. 10.1 g/l dry weight). Thehigher cell densities resulted in an 1.8-fold greater titer of totaltaxanes in the elicitor treatment, i.e., 75.8 mg/L versus 42.4 mg/L forthe control.

The elicitor treatment resulted in increased taxol biosynthesis, both ona per-cell basis (0.098% vs. 0.054% dry weight taxol, a 1.8-foldincrease) and in a titer comparison (13.9 mg/L versus 5.4 mg/L, a2.6-fold increase). The extent of secretion was higher for the elicitortreatment compared to the control (85% versus 72% extracellularproduct).

The elicitor treatment described herein results in increased taxolproduction, a more favorable product profile, enhanced product secretionand retention of high cell viability. These production characteristicsrepresent a significant improvement for a cell culture process for taxolproduction.

Example 9 Production Medium Development

In an effort to increase taxol productivities over the levels describedin example 6, nutrient levels were manipulated to formulate special‘production media’. 7 to 8 day old suspensions of Taxus chinensis lineK-1 grown in Medium D were suction filtered aseptically using a sterileBuchner funnel fitted with a MIRACLOTH (rayon polyester cloth withacrylic binder) filter (Calbiochem). 500 mg fresh weight cells wereaseptically transferred to 5 ml of production Media B and C (see Table2). The vessels were incubated for varying time periods of 18, 25, and42 days at 24±1° C. on a gyratory shaker at 110 rpm in the dark.Treatments were destructively sampled, and observations on growth, colorof the cells and medium, and cell viability were recorded. Samples wereanalyzed for taxanes as described in Example 5. The results of thisexperiment are shown in Table 8.

9.1. Results of 18-day Cultivation

Taxus chinensis cell cultures responded to the altered mediumcompositions by producing significant levels of taxanes and taxol. Thesedata are summarized in Table 9, and a sample chromatogram is shown inFIG. 4. In medium B, 99.8 mg/liter of total taxanes were produced, with24.1 mg/liter of taxol. In Medium C, 110 mg/liter of total taxanes wereproduced, with 21.3 mg/liter of taxol. On a dry weight basis, cellsproduced 0.18% dry weight taxol on medium B, and 0.065% dry weight taxolon medium C.

9.2. Prolonged Cultivation

Taxol and taxane production after prolonged cultivation of Taxuschinensis cells (line K-1) for 25 and 42 days was studied in medium C,the results for which are summarized in FIG. 5. The followingsignificant observations can be summarized:

(i) Taxus suspension cultures are capable of producing significantlevels of taxol and other taxanes. Highest accumulation occurred at 42days, with 0.32% dry weight taxol, and 0.62% dry weight total taxanes;corresponding to titers of 153 mg/L taxol and 295 mg/L total taxanesbased on final medium volume. The analysis of this sample by tandem massspectrometry confirmed the presence of taxol as shown in FIG. 6.Quantitation by MS/MS showed excellent agreement with HPLC.

(ii) The rate of taxol biosynthesis between days 25 and 42 was at ca.7.6 mg taxol per liter per day assuming linear production in the 17-dayperiod. This rate is significantly higher than the rate of production inthe first 25 days. The rate of total taxane biosynthesis between days 25and 42 was 12.3 mg per liter per day. The average volumetricproductivities for taxol, baccatin III, and total taxanes were 3.6, 0.5,and 7.0 mg/L/day respectively.

(iii) Production medium formulations can induce up to 45-fold increasesin specific taxol content compared to rapid-growth conditions (in whichtaxane biosynthesis is unenhanced) such as those described in Example 7.

(iv) The product spectrum can be manipulated so as to funnelbiosynthesis towards the desired end-product taxol, while minimizingproduction of undesirable taxanes. For example, on day 25, taxolconstituted 28% of the total taxanes and on day 42, taxol constituted52% of the total taxanes in contrast to growth medium (see Example 7.1),in which taxol constituted only 12.2% of the total taxanes. This abilityto manipulate product profiles will have important repercussions fordownstream purification and for product purity-related regulatoryissues. For example, the ability to suppress production of the taxaneby-product, cephalomannine could greatly simplify downstreampurification compared to purification of taxol from bark tissue.

(v) Taxus cell cultures have been induced to secrete significant amountsof taxol (87% on day 42) and other taxanes. That the presence ofextracellular taxol and taxanes is due to secretion rather than due tocell lysis is corroborated by several independent observations: (a)Continued biosynthesis occurred between days 25 and 42, suggesting thatcells were viable and active. Independent observations have shownthat >70% viability have been observed after 18 days in productionmedium. (b)

Different percentages of different taxanes were secreted. If cells hadlysed, the percentage in the medium might have been expected to besimilar for the different taxanes.

(vi) The ability of this Taxus cell line to thrive and produce taxol athigh rates in an extracellular environment so rich in product isparticularly worth noting.

(vii) The Taxus cell line with which these results were obtained is alsocapable of rapid growth to high cell densities, and expressed thereported productivities after 20 generations under rapid-growthconditions, attesting to its stability and commercial potential.

The levels of taxol and taxanes produced by cell lines of Taxuschinensis under the conditions described herein are higher thanpreviously reported results by a factor of 35- to 150-fold. For example,Christen et al. (1991) reported the production of 1 to 3 mg/liter oftaxol by suspension cultures of Taxus brevifolia after 2 to 4 weeks ofcultivation. Wickeramesinhe and Arteca (1991) reported the production oftaxol at 0.009% dry weight in cell cultures of Taxus media.

In summary, our data show that with careful initiation and selection ofTaxus chinensis cultures, and with specially formulated growth mediumconditions, cells can be induced to grow rapidly to high cell densities.When these cells are transferred to production medium conditions, cellsare able to biosynthesize and secrete significant levels of taxol andother taxanes for prolonged periods while maintaining high viabilities.The incorporation of periodic medium exchange, light and elicitors withproduction medium results in further synergistic productivityenhancements. These properties are critical prerequisites for anefficient commercial process for taxol and taxane production usingtissue culture technology.

Example 10 10.1. Enhancement of Taxane Production Using Silver

Silver, in the form of silver containing compounds, silver complexes, orsilver ions, was found to be a useful enhancement agent of taxol,baccatin III, and taxane biosynthesis in cell cultures of Taxus species.The combination of silver and other enhancement agents has also beenfound to be useful in obtaining and sustaining high rates of taxaneproduction.

Seven-day old cells of Taxus chinensis suspension KS1A cultivated inMedium L (Table 2) were suction filtered aseptically using a sterileBuchner funnel fitted with a MIRACLOTH (Calbiochem) filter.Approximately 0.75 to 1 gram fresh weight cells were inoculated into 4to 5 mL of culture medium of the given composition indicated in Table10, to yield a fresh weight cell density in the range of 15% to 20%(w/v). The vessels were incubated at 25±1° C. at 120 RPM on a gyratoryshaker (1″ throw) in the dark. Evaporation was corrected for by additionof sterile distilled water. Samples of whole broth (i.e., bothextracellular and intracellular taxanes) were taken at periodicintervals, and were processed and analyzed by HPLC according to themethods outlined in Example 5.

The data summarized in Table 10 indicate that the production of taxol,baccatin III, and other taxanes can be successfully enhanced by avariety of silver containing compounds. This enhancement is dueprimarily to the presence of silver in the medium, as demonstrated inTable 10, which shows enhancement for a variety of different silvercontaining compounds and different counterions. These levels ofproduction are significantly higher than that observed in unenhancedcultures (the production levels for which are elaborated in Example 7).

10.2. Enhancement of Taxane Production using Silver Thiosulfate

Based on considerations of toxicity and ease of preparation and storage,silver thiosulfate was used in subsequent experiments. The method usedfor the preparation of silver thiosulfate was as follows: 1.98 grams ofsodium thiosulfate (pentahydrate) was dissolved in 80 mL of water. 20 mLof a 0.1M solution of silver nitrate was added while stirringvigorously, resulting in 100 mL of a 20 mM stock solution of silverthiosulfate. Potassium thiosulfate could be used in place of sodiumthiosulfate with equally efficacious results. The stock solutions werefilter-sterilized using 0.22 μM cartridge filters into cell culturemedia at the start of a given experiment. Alternative methods forpreparing similar silver thiosulfate solutions are also suitable. Thecell culture protocols were similar to those described for theexperiments described in Table 10.

Table 11 summarizes data obtained by using silver as an enhancementagent for a number of different cell cultures of Taxus chinensis. Thesedata show that silver effects a fundamental enhancement of taxanebiosynthesis generally. The specific product profile observed in anygiven case reflects characteristics of the cell line and the culturemedium. Silver ion/complex can be particularly effective in enhancingtaxane production when used in conjunction with other factors in themedium favoring biosynthesis such as growth regulators, carbon source,salts, micronutrients, and the like.

Example 11 Enhancement of Taxane Production Using Methyl Jasmonate andJasmonate-Related Compounds

The methyl ester of jasmonic acid (methyl jasmonate), as well asjasmonic acid and related compounds, were found to be useful asenhancement agents of taxane biosynthesis in cell cultures of Taxusspecies. The combination of methyl jasmonate and other enhancementagents has also been found to be useful in obtaining and sustaining highrates of taxane production.

Seven-day old cells of Taxus chinensis suspensions cultivated in MediumM (Table 2) were suction filtered aseptically using a sterile Buchnerfunnel fitted with a MIRACLOTH (Calbiochem) filter. Cells wereinoculated into culture medium of the given composition indicated inTable 12, at a fresh weight cell density in the range of 15% to 20%(w/v). The cultures were incubated at 24±1° C. at 120 or 180 RPM(depending on the vessel size) on a gyratory shaker (1″ throw) in thedark. Evaporation was corrected for by adding sterile distilled water.Samples of whole broth (i.e., both extracellular and intracellulartaxanes) were taken at periodic intervals, and were processed andanalyzed by HPLC according to the methods outlined in Example 5.

Table 12 summarizes data obtained by using jasmonic acid and its methylester as enhancement agents for several representative Taxus chinensiscell lines. These data show that jasmonic acid and its methyl estereffect a fundamental enhancement of taxane biosynthesis generally. Thespecific product profile observed in any given case reflectscharacteristics of the cell line and the culture medium. These levels ofproduction obtained in the presence of these enhancing agents aresignificantly higher than that observed in unenhanced cultures (theproduction levels for which are elaborated in Example 7).

Jasmonic acid, its methyl ester, and related compounds, are effectiveenhancement agents of taxane biosynthesis when used in conjunction withother factors in the medium favoring biosynthesis such as otherenhancement agents, growth regulators, carbon source, salts,micronutrients, and the like.

Example 12 Enhancement of Taxane Production Using3,4-Methylenedioxy-6-Nitrocinnamic Acid

The cinnamic acid analog, 3,4-methylenedioxy-6-nitrocinnamic acid (MDNA)and related compounds were found to be useful enhancement agents oftaxane biosynthesis in cell cultures of Taxus species. The combinationof MDNA and other enhancement agents has also been found to be useful inobtaining and sustaining high rates of taxane production.

Seven-day old cells of Taxus chinensis suspension culture SS122-42cultivated in Medium M (Table 2) were suction filtered aseptically usinga sterile Buchner funnel fitted with a MIRACLOTH (Calbiochem) filter.Cells were inoculated into culture medium conditions at a fresh weightdensity of 15% to 20% (w/v). The vessels were incubated at 24±1° C. at180 RPM on a gyratory shaker (1″ throw) in the dark. Treated cultureswere sampled and analyzed using the methods described in Example 5 atvarious time points. Evaporation was corrected for by adding steriledistilled water at periodic intervals. Samples of whole broth (i.e.,both extracellular and intracellular taxanes) were taken at periodicintervals, and were processed and analyzed by HPLC according to themethods outlined in Example 5.

Table 13 summarizes data obtained by using3,4-methylenedioxynitrocinnamic acid as an enhancement agent for taxanebiosynthesis in Taxus chinensis cell cultures. These data show that MDNAeffects a fundamental enhancement of taxane biosynthesis generally.Cultivation in Medium II i.e., in the presence of MDNA and silver,further enhances the production of taxanes. The specific product profileobserved in any given case reflects characteristics of the cell line andthe culture medium. These levels of production are significantly higherthan that observed in unenhanced cultures (the production levels forwhich are elaborated in Example 7).

Example 13 Enhancement of Taxane Biosynthesis Using a Combination ofEnhancement Agents

Various enhancement agents, used in combination, gave significant andsynergistic improvements in taxane production.

Seven-day old cells of Taxus chinensis suspension cultures cultivated inMedium P(SS64-412), Medium O (SS64-561, SS64-571), Medium I (SS124-77,SS85-26), Medium M (SS122-29) (the composition of these media are listedin Table 2) were suction filtered aseptically using a sterile Buchnerfunnel fitted with a MIRACLOTH (Calbiochem) filter. Cells wereinoculated into culture medium (indicated in Table 14) at a fresh weightdensity of 20% (w/v). The cultures were incubated at 24±1° C. at 180 RPMon a gyratory shaker (1″ throw) in the dark. Evaporation was correctedfor by adding sterile distilled water at periodic intervals. Samples ofwhole broth (i.e., both extracellular and intracellular taxanes) weretaken at periodic intervals, and were processed and analyzed by HPLCaccording to the methods outlined in Example 5.

Table 14 summarizes data obtained by using various combinations ofenhancement agents for taxol, baccatin III, and taxane biosynthesis inTaxus chinensis cell cultures. The data demonstrates substantial furtherenhancement of taxane production by combinations of enhancement agentsover that seen for individual agents, and over production levels inunenhanced conditions (the production levels for which are elaborated inExample 7).

Example 14 Enhancement of Taxane Production by Medium Exchange

This example demonstrates that high productivity in culture can besustained by replenishing medium components and removing spent medium.

Cell lines were initially cultivated in Medium O (Paella), Medium I(SS29-3A5), and Medium I(SS45-146). The detailed compositions of thesecultivation media are described in Table 2. Seven day-old cells of thesecell lines were suction-filtered aseptically using a sterile Buchnerfunnel fitted with a MIRACLOTH (Calbiochem) filter. Approximately 1.5grams fresh weight cells were inoculated into 4.25 mL of the respectiveculture media indicated in Table 15. The vessels were incubated at 24±1°C. at 120 RPM on a gyratory shaker (1″ throw) in the dark. Evaporationwas corrected for by addition of sterile distilled water at periodicintervals. For the medium exchange treatments, the spent productionmedium was suctioned off using a sterile pipette after 10 to 11 days ofbatch cultivation, leaving the cells behind in the vessel. The spentsupernatant was analyzed for extracellular taxanes using the methodsdescribed in Example 5. Fresh culture medium of the same composition asthe first batch culture was added to the vessel containing productivecells. The cells were cultured under the same environmental conditionsdescribed above. The medium exchange cycle was repeated after anadditional 10 to 11 days of cultivation. The total extracellular taxanesfor batch production is compared with that of medium exchange productionin Table 15. The medium exchange concentration values denote the totalamount of taxane produced in the extracellular medium divided by thevolume of the cell suspension culture (i.e., 5.75 mL).

Table 15 indicates that cells can be sustained in a productive state fora prolonged period, and in fact, that productivity of the cells can beenhanced by repeated medium exchange. Enhancement by repeated mediumexchange is feasible using a range of different enhancement conditions,and with a variety of cell cultures.

The data demonstrates substantial further enhancement of taxaneproduction over production levels in unenhanced conditions (theproduction levels for which are elaborated in Example 7).

Example 15 Enhancement of Taxane Production by Fed Batch Operation

Seven day-old cells of cell lines cultivated in Medium I (CR-128,SS36-245), Medium L (SS36-359) (the compositions of these media aredescribed in Table 2) were suction filtered aseptically using a sterileBuchner funnel fitted with a MIRACLOTH (Calbiochem) filter.Approximately 1 gram fresh weight of cells were inoculated into 4 ml ofculture medium of the given composition indicated in Table 16.a. Thevessels were incubated at 24±1° C. at 120 RPM on a gyratory shaker (1″throw) in the dark. Evaporation was corrected for by addition of steriledistilled water at periodic intervals. For fed batch operation, sterilefeed solutions of predetermined compositions were fed continuously intothe culture vessels at predetermined rates of feeding, e.g. 10 mL feedsolution per liter of culture per day. Details of the fed batchoperation are described in Table 16.b., including compositions of thefeed solutions and feeding protocols. Treated cultures were sampled andanalyzed using the methods described in Example 5.

Table 16.a. indicates that cells can be sustained in a productive statefor a prolonged period, and in fact, that productivity of the cells canbe enhanced by fed batch operation, resulting in the accumulation ofhigh levels of baccatin III, taxol, and other taxanes. The relativeamounts of particular taxanes reflect the interaction of feedingprotocol and feed composition with the cell line and culture conditions.This Table also indicates that feeding phenylalanine results in enhancedproduction of taxol relative to other taxanes.

The data demonstrates substantial further enhancement of taxaneproduction over production levels in unenhanced conditions (theproduction levels for which are elaborated in Example 7).

Example 16 Enhancement of Taxane Biosynthesis Using a Combination ofEnhancement Agents

Various enhancement agents, used in combination, gave significant andsynergistic improvements in taxol, baccatin III, and taxane production.

Seven-day old cells of Taxus chinensis suspension cultures (SS122-41,cr427, SS122-30, cr857, cr452) cultivated in Medium M (the compositionof the medium is listed in Table 2) were suction filtered asepticallyusing a sterile Buchner funnel fitted with a MIRACLOTH (Calbiochem)filter. Cells were inoculated into culture medium (indicated in Table17) at a fresh weight density of 20% (w/v) unless described otherwise inTable 17. The cultures were incubated at 24±1° C. at 180 RPM on agyratory shaker (1″ throw) in the dark. Evaporation was corrected for byadding sterile distilled water as necessary. Samples of whole broth(i.e., both extracellular and intracellular taxanes) were taken atperiodic intervals, and were processed and analyzed by HPLC according tothe methods outlined in Example 5.

Table 17 summarizes data obtained by using various combinations ofenhancement agents for taxol and taxane biosynthesis in Taxus chinensiscell cultures. The data demonstrates substantial further enhancement oftaxane production by combinations of enhancement agents over that seenfor individual agents, and over unenhanced conditions (the details ofwhich are provided in Example 7).

Example 17 Enhancement of Taxane Production by Fed Batch Operation

Seven day-old cells of cell lines cultivated in Medium M (SS122-41) (thecompositions of these media are described in Table 2) were suctionfiltered aseptically using a sterile Buchner funnel fitted with aMiracloth (Calbiochem) filter. Approximately 1 gram fresh weight ofcells were inoculated into 4 ml of culture medium of the givencomposition indicated in Table 18.a. The vessels were incubated at 24±2°C. at 120 RPM on a gyratory shaker (11 throw) in the dark. Evaporationwas corrected for by addition of sterile distilled water. For fed batchoperation, sterile feed solutions of predetermined compositions were fedcontinuously into the culture vessels. Details of the fed batchoperation, including compositions of the feed solutions and feedingprotocols are described in Table 18.b. Treated cultures were sampled andanalyzed using the methods described in Example 5.

Table 18.a. indicates that cells can be sustained in a productive statefor a prolonged period, and in fact, that volumetric productivity of thecells can be enhanced by fed batch operation, resulting in theaccumulation of high levels of baccatin III, taxol, and other taxanes.The relative amounts of particular taxanes reflect the interaction offeeding protocol and feed composition with the cell line and cultureconditions.

The data demonstrates substantial further enhancement of taxaneproduction over production levels in unenhanced conditions (theproduction levels for which are elaborated in Example 7).

For purposes of clarity of understanding, the foregoing invention hasbeen described in some detail by way of illustration and example inconjunction with specific embodiments, although other aspects,advantages and modifications will be apparent to those skilled in theart to which the invention pertains. The foregoing description andexamples are intended to illustrate, but not limit the scope of theinvention. Modifications of the above-described modes for carrying outthe invention that are apparent to persons skilled in the art areintended to be within the scope of the invention, which is limited onlyby the appended claims.

All publications and patent applications mentioned in this specificationare indicative of the level of skill of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

TABLE 1.a. List of Elicitors Used in Elicitation of Taxus spp. CellCultures I. Biotic Elicitors (microorganisms) Botrytis cinereaPhytophthora megasperma Pinellas stripticum Oligosporus sp. Pythiummamillatum Pythium sylvaticum Verticillium dahliae Verticillium sp.Penicillium minioluteum Phytophthora lateralis Cytospora cinctaCytospora leucostoma Alternaria brassicicola Alternaria solaniAlternaria cucumerina Botrytis squamosa Cochliobolus heterostrophusColletotrichum trifolii Colletotrichum orbiculare Colletotrichumgraminicola Colletotrichum gloeosporioides Cylindrocladium floridanumFusarium crookwellense Fusarium heterosporium Fusarium oxysporum f. sp.conglutinans Fusarium oxysporum f. sp. lycopersici Fusarium oxysporum f.sp. pisi Gibberella zeae Gaeumannomyces graminis var. tritici Geotrichumsp. Leptosphaeria korrae Nectria haematococca MPVI Mycosphaerellapinodes Ophiostoma ulmi Phoma lingam Phoma pinodella Phytophthorainfestans Pythium aristosporum Pythium graminicola Pythium ultimumRhizoctonia solani Sclerotinia sp. S. nodorum D-45 Trametes versicolorUstilago maydis Venturia inaequalis II. Biotic Elicitors (Microbialfractions or products) Chitosan Cellulysin Lichenan Multifect XLGlucomannan Multifect CL Pleuran Resinase Glucan PulpxymeCarboxymethylglucan SP431 Hydroxymethylglucan Pectinol SulfoethylglucanRapidase Mannan Klerzyme Xylan Chitinase Mannobiose MannotrioseMannopentaose Mannotetraose III. Abiotic Elicitors (Chemical StressAgents as well as some naturally occurring biochemicals) Arachidonicacid Elaidic acid Cyclic AMP Dibutyryl Cyclic AMP Methyl jasmonateCis-Jasmone Miconazol Ferulic acid AMO-1618 Triton X-100 Benzoic acidand derivatives Salicylic acid and derivatives Propyl gallate SesamolChlorocholine chloride 3,4-dichlorophenoxy triethyl (amine)Chloroethylphosphonic acid Diethyldithiocarbamic acidNordihydroguaiaretic acid Sodium metabisulfite Dithiothreitolb-amino-DL-Phenylalanine Potassium metabisulfite Uniconazol Vanadylsulfate Spermine Paclobutrazol Putrescine Spermidine CadavarineProtamine Sulfate SKF-7997 MER 29 Ancymidol Triadimefon Phosphon DThiourea Dextran Sulfate Hydroquinone Chitosan glutamate FenpropemorphProchloraz Naptifine EDU HTA MPTA Glutathione EGTA Gibberellins AbscisicAcid 1,3-Diphenyl urea Diazolidinyl urea Phloroglucinol Sodium alginateCarragenan

TABLE 1.b List of Precursors, Inhibitors & Stimulants or Activators Usedin Regulation of Biosynthesis of Taxol & Taxanes in T. spp. cellcultures. Precursors Inhibitors Stimulants Phenylalanine Chlorocholinechloride Cyclic AMP Lysine Uniconazol Dibutyryl Cyclic AMP TyrosinePaclobutrazol Methyl jasmonate Tryptophan SKF-7997 Cis-JasmoneMethionine MER 29 Chloroethylphosphonic acid Tyramine Ancymidol SpermineAcetic acid and Triadimefon Spermidine its' salts Phosphon D PutrescineMevalonic acid Fenpropemorph Cadavarine Farnesyl acetate Prochloraz MPTAGeranyl acetate Naptifine DCPTA Geranylgeraniol acetate Miconazol DIPTATryptamine Silver Nitrate ACC Menthol Norbornadiene HTA a-Pinene AMO1618 Brassinosteroids Trans-cinnamic acid Alar BHA Cambrene A4-amino-5-Hexynoic acid BHT Verticillene Phenylethanolamine OTAVerticillol Phenethylamine Camphor Glyphosate QuercetinDihydrocycloeucalenol Levulinic acid Methionine Sulfoxide Abietic acidβ-Hydroxyphenethylamine Borneol 5-Methyl-DL-Tryptophana-Fluorophenylalanine 5-2 Aminoethyl-L-cysteine hydrochloride

TABLE 1.c ELICITORS Xylanase Butaclore ChitooligosaccharidesButylisothiocynate Spermine Bis Nitric oxide Adduct ChlorambenN,N′-Diacetylchitobiose isopropylamine Ethyl carbamate Bis2-Hydroxyethylhydrazine Nitric oxide Adduct Hydroxyglutaric aciddisodium Diethylamine Bis (Nitric oxide) Adduct Tryptophol BenzylN,N′-Diacetyl-β-chitobioside Thiourea Syringic acid ThioacetamideBenzothiadiazole 2,4,6-Trichlorphenol Bipyridyl Pyridine-2-aldoximeGossypol and derivatives methochloride 2-chlor-4-methylisonicotinic acidPotassium oxalate monohydrate Indomethacin Poly-L-Lysine hydrobromideN,N′,N′-Triacetylchitotriose Nerol N,N′-DiacitylchitobioseN-(1-Naphthyl) phthalamic acid Diammoniun oxalate Oxalate NigeranOctapomine hydrochloride p-hydroxyacetophenone Oxizamide Pectic acid2-Methylpyrazine Lysozyme Methoxyacetic acid Nitric oxideN-Ethoxycarbonyl-2-ethoxy-1,2- Glutathione (reduced) Dihydroquinoline1,2-Diaminopropane Lanthanum acitate 1,3-Diaminopropane Linolenic acidβ-mercaptoethylamine Lipase Hydroxylamine Iodoacetamide Deoxyglucose2-hydroxyethylhydrazine 2-chlorobenzoic acid Dinocap 2-Methyl-1,2-DL(3-Pyridyl) 1-Propane 1,3-Diphenylurea 5-Bromouracil Hydrogen peroxide7-Nitrondazole Urea hydroperoxide 8-Hydroxyquinoline Sebacic acidAcedoamidocinnamic acid Benzoyl peroxide 2-AminoanthraquinoneN-methylmaleimide N-Acetyl-L-glutamic acid Cumen peroxide AgmatinN-Acetyl-D-Glucosamine 3-Acetyl pyridine Octyl-β-D-GlucopyranosideButyryl Butyryl Lactate Diisopropyl fluorophosphate7-Bromo-5-chloro-8-hydroxyquinoline Isopropyl-β-D- Benzylbenzoatethiogalactopyranoside Bromoxynil Hydroxyethyl-β-1,3-glucanSyringaldehyde Dextran Chitinase Lucifer yellow Bacitracin Calciumcyanide Glucans Glutaric acid Morpholine OctamethylcyclotetrasiloxaneTrigonelline hydrochloride Anthranilic acid Colistin methane sulfonateColchicine 2,4-Dichlorophenol L-Phenylalanine-2-naphthylamideHydroxyglutaric acid, and its salts DL-2-Hydroxy-3-methylbutyric acid1-10-Phenanthroline monohydrate N-sulfosuccinimidyl-3-(4-Hydroxyphenyl)propionate Trans-1,6-diphenylhexatriene Arachidonic acidUrea hydrogen peroxide Hydrogen peroxide Bestatin Butylatedhydroxyanisole Butylated hydroxytoluene Gellan gum cellulase Pimelicacid Diisopropyl phosphochloridate Nitrapyrin t-Butyl hydroperoxideDL-Phosphinothricin ammonium Methyl syringate Trifluralin TridecanoneMimosine Narigenin Dimethylaminopyridine 1-BenzylimidazoleDL-o-chlorophenylalanine Cetylpyridinium chloride HydroquinoneSyringomycin

TABLE 1.d PRECURSORS Dimethylphenylalanine D-fructose-1,6-DiphosphateGeranyl chloride β-Hydroxypyruvic acid Geranylgeraniol4-Hydroxyphenylpyruvic acid trans-Cinnamic acid Methyl acetate Pyruvicacid Methyl laurate Phenylpyruvic acid Oxaloacetic acidOrthosuccinylbenzoic acid Pinenes 2,3-dihydrobenzoic acid Geranylacetate o-hydroxyphenylpyruvic acid Nerol Postassium acetatePhellandrene Glutamic acid Benzoyl chloride Aspartic acid R(−)Citramalicacid DL-β-phenylserine Aspargine Hippuric acid 2,3-Dichlorobenzoic acidp-Hydroxycinnamic acid Isoleucine Benzyl acetate Leucine Phenylaceticacid Phosphoglyceric acid 3-Benzoylpropionic acid Serine Citric acid2-Hydroxycinnamic acid Calcium benzoate 3-Hydroxycinnamic acid Arginine4-Hydroxycinnamic acid N-Benzoyl-DL-Phenylalanine Borneol3,4-Dihydroxycinnamic acid Phosphoglycerate Potasium SaltPhosphoenolpyruvic acid Glyceraldehyde-3-phosphate PhenylisoserineDihydroxyacetone phosphate 4-Hydrocoumarin Glycine Glutamine Ethylacetate Ornithine Methyl cinnamate Methionine Potassium acetate Shikimicacid DL-Glyceraldehyde-Phosphate free acid Oxoglutamic acid Calciumbenzoate DL-3-Amino-3-phenylpropionic acid Oxoglutamic acida-Phenylalanine Phosphoenolpyruvic acid β-Phenylalanine MentholN-Benzoylphenylisoserine Cambrene A Geraniol Verticillol LinaloolVerticellene Geranyl linalool Abietic acid Isoborynyl isovalerateSuccinic acid Cinnamyl acetate Fumaric acid Cinnamyl propionateAcetoacetate Potasium Salt Cinnamyl chloride

TABLE 1.e INHIBITORS Rhizobitoxine Trans-3,4-difluorocinnamic acida-Canaline Mercaptoethanol a-Aminosobutyric acid 4-Hydroxycoumarincis-Propenylphosphonic acid Cinnamulfluorene Flurprimidol2-Cyano-4-Hydroxycinnamic acid Chloromethyl CyclopropaneCinnamylidenemalonic acid Diazocyclopentadiene 4-Dimethylaminocinnamicacid Diammonium succinate N-Cinnamylpiperazine g-GlutamylmethylamideN-trans-Cinnamoylimidazole 2,3-Dimercaptosuccinic acidCinnamylideneacetophenone p-Nitrophenylphosphate 3,4-Methylenedioxycinnamic acid Pervanadate 3,4-Methylenedioxy-6-nitrocinnamicOrthovanadate acid N-Acetyl-DL-homocysteine Thiolactone3-(3,4-Methylenedioxyphenyl)propionic 2,3-diphosphoglyceric acid saltsacid p-Hydroxymercurylbenzoate 3,4-Methylenedioxyphenylacetic acidMethylmercury chloride 3,4-trans-Dimethoxycinnamic acidMethylcyclopropane 4-Methoxycinnamic acid Methylcyclopropane carboxylate2-Methoxycinnamic acid Cyclooctodine 4-Nitrocinnamic acid ethyl esterMethoxyvinyl glycine Methoxycinnamic acid Ibuprofen4-Nitrocinnamaldehyde Piperonylic acid 3-Nitrocinnamic acidPhenylpropiolic acid 2-Nitrocinnamic acid L-2-Hydroxy-3-phenylpropionicacid 3,4-Dimethoxy-6-nitrocinnamic acid Amino oxyacetic acid Ammoniumoxalate D-Phenylalanine Sinapic acid Phenylpyruvic acid2-Hydroxy-4,6-dimethoxybenzoic acid L-Tyrosine 3-dimethylaminobenzoicacid 4-Fluoro-(1-amino-2-phenylethyl)Phosphonic 3,4-dimethoxybenzoicacid acid 4-Methoxybenzoic acid 4-Hydroxyphenylpyruvic acidN(G)-Nitro-D-Arginine m-Fluoro-DL-phenylalanine N(G)-Nitro-L-Argininep-Fluoro-DL-phenylalanine Malonic acid m-Fluoro-DL-tyrosine Maleic acidhydroxide 3,4-Difluoro-D-phenylalanine Okadaic acid 1-Aminobenzotriazol1,4-Cyclohexanedione 4-Fluorocinnamic acid Diisopropyl fluorophosphateSKF-525A Oxamic acid Diethyldithiocarbamic acid, Sodium Salt Oxamicacid, derivatives Dithiothreitol Sulfanilamide p-Coumaric acidN-Acetyl-S-farnesyl-L-cysteine Vinylimidazole Chaetomellic acid A,sodium salt a-Hydroxyfarnesylphosphonic acid Isonicotinic acid hydrazideN6-Monomethyl-L-arginine 2,3-dimercaptopropanol 7-NitroondazoleSalicylhydroxyamic acid Norflurazon 3-amino-4-hydroxybenzenesulphonicCyclooctodieneα-Fluorophenylalanine acid Diethyldithiocarbamic acidHydroxyurea SKF-7997[Tris-(2-diethylaminoethyl)-6,7-dimethoxy-1,2-benzisoxazole-3-acetic phosphate trichloride] acidTriadimefon 3-oxo-1,2-benzisothiazoline-2-ylacetic2,3,4-Trimethoxycinnamic acid acid 2,4-Dimethoxycinnamic acid2,3,5-Triidobenzoic acid 3-Hydroxyphenylacetic acid2-(p-Chlorophenoxy)-2- 4-Aminotriazole methylpropionic acid4-Fluorocinnamic acid N-(1-Naphthyl)phthalamic acid4-Chloro-2-methylphenoxyacetic acid 1-Pyrenoxylbenzoic acid1,3-Dichloropropane 2-Chloro-9-hydroxyfluorene-9-carboxylicN-Ethylmaleimide acid Semicarbizide Chlorocholine chloride4-Chlororesorcinol 2′-Isopropyl-4′-(trimethylammonium1,2-Dichloropropane chloride)-5-methyl phenylpeperidone Idoacetamidecarboxylate Phenylhydrazine Sesamol Silver thiosulfate Ancymidol Silverchloride Daminozide Thiosemicarbazide LovastatinN-(phosponomethyl)-Glycine Simvastatin p-Chlorophenoxyisobutyric acidCaffeic acid Triton x-100 Ferulic acid Triparanol 2,5-Dihydroxycinnamicacid Chlorphonium chloride 2,5-Dihydromethoxycinnamic acid Mepiquat4-Hexylresorcinol Prohexadione calcium salt Cetylpyridinum chlorideChloromequat Stourosporine Tetcyclasis Dimethylthiourea2-Aza-2,3-dihydrosqualene Phenylpropiolic acid Dinoconazole Ammoniumoxalate Tridemorph 1-Aminobenzotriazole 2,3-Iminosqualene1-Vinylimidazole Glyphosine Mercaptoethanol Isoprophyl-N-phenylcarbamate 3,5-Diido-4-hydroxybenzoic acid Oryzalin5-Methyl-7-chloro-4-ethoxycarbanylmethoxy- Caffeine2,1,3-benzothiadiazole D-Arginine Bromoxynil α-Methylornithine3,4,5-Trichlorophenol Conavanine N-Methylmaleimide Abscisic acid4-Fluoro-DL-tyrosine 3-Amino-1,2,4-triazole Ethyl-3-nitrocinnamate4-Nitrocinnamic acid Conavanin 3,4-Dimethoxyphenylacetic acidMethylacetylenic putrescine N-Cinnamylpiperazine Methylpyruvic acidHydroxylamine α-Hydroxy-2-pyridinemethane sulfonic2,4-Dinitrophenylhydrazine acid Tetramethylammonium bromideAcetohydroxamic acid Clotrimazole Isopropyl-N-phenyl carbamateValinonycin D1-phenylene iodonium Procaine 2-Aminoindan-2-phosphonicacid Monensin Potassium-arsenate Uniconazoleα-aminooxy-β-phenylpropionic acid Paclobutrazole Benzyl hydroxylamine4-Aminotriazole Piperonyl butoxide Benzyl isothiocyanateSelenomethionine 1-Acetyl-2-thiourea 3,4-Dehydro-DL-proline2-Ethylnaphthalene 3-Nitrobenzoic acid Silver salts such as Silverchloride, Silver nitrate, etc. Sodium hydrosulfite 7-nitronadozoleEthionine Azacytididine Ethoxy-carbonyl-pyrimidine Miconazole2,3:4,6-Di-o-isopropylidene-2-keto-L-Gulonic acidN-(4-Hydroxyphenyl)glycine 3-(4-Hydroxyphenyl)propionic acid3-(2-Hydroxyphenyl)propionic acid 4-CyclohexanedioneN-(6-aminohexyl)-5-chloro-1-Naphthalenesulfonamide hydrochlorideEndothal Phosphan Cyanamide α-(1-Methylethyl)-α-(4-trifluoromethoxy)phenyl-5- pyrimidinemethanol 2-Aminoisobutyric acidD-Arginine n-Butylamine p-Chloromercurybenzene sulphonic acidMethylglyoxal bis (guanyl hydrazone) α-Methyl ornithine

TABLE 1.f STIMULANTS Potassium pyrophosphate p-aminohippuric acid Sodiumpyrophosphate Benzylcinnamate Uracil Jasmonic acid Melatonin Methyljasmonate Hydroxylamine hydrochloride Dihydroisojasmone ThionicotinamideIsojasmone S-adenosyl-L-methionine cis-jasmone Inosine triphosphateTetrahydrojasmone Indole-3-lactic acid Lactone of cis-jasmoneIndole-3-pyruvic acid Dihydrojasmone Indole-2-carboxylic acidJasminolactone Indole-3-aldehyde Jasmolactone N-indolyl acetyl valine12-oxophytodienoic acid Pyridoxal phosphate Jasmonol Methyldihydrojasmonate g-methyldecalactone Bipyridyl Citronellyl tiglate4-acetamidophenol Jasmonyl acetate Imidazole MastoparanOctyl-β-D-glucopyranoside Lysophosphatidic acid 3-aminopyridineCypermethrin Guanylic acid Cantharidin Citydylic acid Acetylsalicylicacid Isopropyl-β-d-thiogalactopyranoside Salicylic acid and derivatives3-(4-hydroxyphenyl) propionic acid 2,6-dichloroisonicotinic acid3-(2-hyroxyphenyl) propionic acid Nitric oxide Indole-3-pyruvic acidTraumatic acid Thiobenzoic acid Citric acid DimethylaminophenylalanineCytidylic acid p-hydroxyphenylpyruvic acid malic acid or malic acid salt2,3-dihydroxybenzoic acid Potassium malate Ethyl benzoate Citric acidsalts and derivatives 3,4-dihydroxycinnamic acid Flavin adeninemononucleotide 4-hydroxycinnamic acid Flavin monocleotideN-acetyl-L-phenylalanine dibutyrl Cyclic AMP 3-Benzoylpropionic acidSpermine p-hydroxycinnamic acid Spermidine 5′,5′-Dithiobis(2-nitrobenzoic acid) Putrescine β-hydroxypyruvic acid Cadavarine4-hydroxyphenylpyruvic acid S-Adenosylmethionine Methyl cinnamatePyridoxal phosphate Methyl salicylate 6-Aminonicotinamide2-napthylbenzoate 4-Dimethylaminopyridine PhenylsalicylateN-(2-Hydroxyethyl)succinimide Thiosalicylic acid 2-oxoglutaric acidPropachlor Thiamine Vinyl propionate Triethylamine hydrochloride3,5-Diisopropylsalicylic acid Adenine sulfate p-Amino-L-PhenylalanineBenzyl salicylate 1,2-Benzisoxazole 2,4-Carbonyldibenzoic acidL-Citrulline D-Erythrose 4-Phosphate Fructose 1,6-Diphosphate Inosinetriphosphate N-Methylputrescine dihydrochloride β-Phenylethylaminehydrochloride Lysine Imidazole Guanylic acid MelatoninAminocyclopropane-carboxylic acid IsopentylpyrophosphateN-Acetyl-L-glutamine Isoglutamine Threonine Potassium PyrophosphateSodium pyrophosphate L-2-Aminoadipic acid N-methyl-N-Propagylbenzylaminehydrochloride Aminoguanidine hemisulfateL-(+)-2-Amino-7-Phosphonoheptanoic acid Ammonium sulfamate Spermine BisNitric oxide adduct Diethylamine Bis Nitric oxide adduct GalactoseValine Vitamin B-12 Ascorbic acid and derivatives CoronatinePhenobarbital Pregnenolone 24-epi-Brassinolide n-Propyl DihydrojasmonatePropyl jasmonate Epimethyl jasmonate

TABLE 2 Composition of media used for cultivation of Taxus speciescultures. Medium A B C D E F G H I J K L M N O P Chemical Ingredientmg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/L mg/Lmg/L mg/L Ammonium Nitrate 400.0 500.0 400.0 Ammonium Sulfate 134.0 33.5134.0 67.0 134.0 134.0 134.0 134.0 134.0 134.0 33.50 134.0 134.0 BoricAcid 3.0 1.5 0.75 3.0 1.5 0.75 6.2 1.5 3.0 3.0 3.0 3.0 3.0 0.75 3.0 3.0Calcium Chloride 113.24 28.31 113.24 56.62 72.5 113.24 72.5 113.24113.24 113.24 113.24 113.24 28.31 113.24 113.24 (anhydrous) CalciumChloride 20.0 50.0 50.0 2-H2O Calcium Nitrate 206.4 366.0 366.0 4-H2OCobalt Chloride 0.026 0.006 0.025 0.0125 0.025 0.025 0.025 0.025 0.0250.025 0.01 0.025 0.025 6-H2O Cupric Chloride H2O 0.01 Cupric Sulfate5-H2O 0.025 0.01 0.006 0.025 0.0125 0.25 0.025 0.25 0.025 0.025 0.0250.025 0.025 0.01 0.025 0.025 Na2 EDTA 2-H2O 37.3 9.32 37.3 18.65 37.337.3 37.3 37.3 37.3 37.3 37.3 37.3 0.33 37.3 37.3 Ferric Sulfate 2.5Ferrous Sulfate 7-H2O 27.85 6.95 27.85 13.9 27.85 27.85 27.85 27.8527.85 27.85 27.85 27.85 6.96 27.85 27.85 Magnesium Sulfate 122.09 366.230.5 122.09 61.04 180.7 122.09 180.7 122.09 122.09 122.09 122.09 122.0930.52 122.09 122.09 (anhydrous) Manganese Sulfate 10.0 23.788 22.5 10.05.0 22.3 10.0 22.3 10.0 10.0 10.0 10.0 10.0 27.60 10.0 10.0 H2OMolybdenum Trioxide 0.001 Molybdic Acid 0.25 0.062 0.25 0.125 0.25 0.250.25 0.25 0.25 0.25 0.25 0.25 0.06 0.25 0.25 (sodium salt) 2-H2OPotassium Chloride 65.0 Potassium Iodide 0.75 0.75 0.175 0.75 0.375 0.750.75 0.75 0.75 0.75 0.75 0.19 0.75 0.75 Potassium Nitrate 2500.0 80.0625.0 2500.0 1250.0 2500.0 2500.0 2500.0 2500.0 2500.0 2500.0 625.002500.0 2500.0 Potassium Phosphate 10.0 170.0 170.0 (monobasic) PotassiumSulfate 990.0 990.0 Sodium Phosphate 130.5 16.5 32.62 130.5 65.25 130.5130.5 130.5 130.5 130.5 130.5 32.63 130.5 130.5 (monobasic anhydrous)Sodium Sulfate 200.0 Zinc Sulfate 7-H2O 2.0 3.0 0.5 2.0 1.0 8.6 2.0 8.62.0 2.0 2.0 2.0 2.0 0.50 2.0 2.0 Myo-inositol 100.0 100.0 125.0 100.050.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 100.0 25.00 100.0 100.0Nicotinic Acid 1.0 0.75 1.0 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 0.25 1.01.0 Pyridoxine HCL 1.0 0.25 1.0 0.5 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.251.0 1.0 Thiamine HCL 10.0 *5.0 3.5 10.0 5.0 10.0 10.0 10.0 10.0 10.010.0 10.0 10.0 2.50 10.0 10.0 Glutamine 292.8 146.4 292.8 292.8 1756.8292.8 292.8 292.8 292.8 292.8 292.8 Tryptophan Phenylalanine 30.0 Lysine20.0 Methionine Sodium Acetate 10.0 10.0

ucrose 10000.0 50000.0 40000.0 10000.0 10000.0 10000.0 20000.0 10000.010000.0 10000.0 10000.0 10000.0 50000.0 10000.0 10000.0

Benzyladenine 0.002 2.0 2.0 0.002 0.002 0.002 0.002 0.02 0.02 0.002 0.02

Naphthaleneacetic 0.931 10.0 1.862 0.931 0.931 1.862 1.862 0.931 1.862Acid Ascorbic Acid 50.0 100.0 50.0 100.0 100.0 100.0 100.0 100.0 100.0100.0 100.0 100.0 100.0 100.0 100.0

choram 1.2 2.4 1.2 1.2 2.4

asein Hydrolysate 500.0 1000.0

-Dimethyl- 0.02 allylamino) Purine

inatin 0.02

hidiazuron 0.022

altose 10000.0 Glutamic Acid 1850.0 1850.0 1850.0 1850.0 Aspartic Acid1710.0 Glycine 5.0 Serine 5.0 Folic Acid 1.0

odium pH 5.6 5.8 5.8 5.6 5.6 5.6 5.6

5.6 5.6 5.6 5.6 5.6 5.8 5.6 5.6 Indicates that the

indicates data missing or illegible when filed

TABLE 3 Preferred conditions for callus proliferation for various Taxusspecies. The ingredients in the basal media are listed in Table 2. BasalGrowth Regulators* Medium Auxin Cytokinin Species (Table 2) Type Conc(M) Type Conc (M) T. brevifolia F P 5 × 10−⁶ 2iP 10−⁷ D P 5 × 10−⁶ BA10−⁸ T. canadensis H P 5 × 10−⁶ K 10−⁷ D P 5 × 10−⁶ BA 10−⁸ T. chinensisD P 5 × 10−⁶ BA 10−⁸ A N 5 × 10−⁶ BA 10−⁸ T. globosa D P 5 × 10−⁶ BA10−⁸ T. floridana D P 5 × 10−⁶ BA 10−⁸ T. baccata D P 5 × 10−⁶ BA 10−⁸T. cuspidata D P 5 × 10−⁶ BA 10−⁸ T. media D P 5 × 10−⁶ BA 10−⁸ T.wallichiana D P 5 × 10−⁶ BA 10−⁸ *Abbreviations: Picloram (P),Naphthalene acetic acid (N), Benzyladenine (BA), Dimethyl allylaminopurine (2iP), Kinetin (K)

TABLE 4 Typical growth characteristics of Taxus sp. suspension culturesDry Weight Fresh Weight Fresh Doubling Doubling Dry Wt. Wt. Species TimeTime Density Density T. brevifolia 2.0 days 3.5 days 20 g/L 400 g/L T.baccata 2.0 6.0 15 220 T. chinensis 2.5 4.5 20 285 T. canadensis nd* 8.513 260 *not yet determined

TABLE 5 Taxol production in various Taxus species. Taxol content MediumSpecies (% dry weight) (See Tables 2 & 3) Analysis T. brevifolia 0.006 FELISA T. canadensis 0.004 H ELISA T. baccata 0.0014 D HPLC T. globosa0.0003 G ELISA T. cuspidata 0.0025 G HPLC T. floridana 0.001 G ELISA T.media 0.02 F ELISA T. chinensis 0.18 B HPLC

TABLE 6 Improvements in productivity due to medium exchange treatment.Numbers are expressed as X-fold improvement over levels achieved in a15-day batch interval. Taxus chinensis cell line K-1 was cultivated inMedium A in the dark. Total levels* Extracellular levels Taxol 4.6 4.89Total taxanes 4.55 5.94 *Total levels in cells and medium combined

TABLE 7 Effect of Standard GroLux light treatment on taxol and taxanecontent in 10-day old cultures of Taxus chinensis line K-1 cultivated inMedium A. Amounts shown are expressed as mg extracted from 20 ml ofsuspension. Cell growth was identical in both treatments (164 mg dryweight per flask). Light Dark Total taxol: cells and medium:  8.8 μg 3.13 μg Extracellular taxol: 76.40% 56.20% Total taxanes cells andmedium: 61.55 μg 62.17 μg Extracellular taxanes:   89%   84%

TABLE 8 Comparison of chitosan-glutamate treated to non-elicitedsuspensions of Taxus chinensis line K-1 after 15 days cultivation inmedium C. Taxane levels reported are from cells and medium combined. %extra refers to the percentage of extracellular CONTROL ELICITOR Celldensity 10.1 g/L Cell density 14.2 gm/l Cell viability 70-80% viableCell viability 75-80% viable % % % % Taxanes dry wt mg/L Extra dry wtmg/L Extra Taxol 0.054 5.4 7.2 0.098 13.9 85.0 Baccatin III 0.057 5.869.9 0.055 7.8 76.6 7-Xylosyl-10- 0.040 4.0 63.0 0.048 6.9 77.0deacetyltaxol 10-deacetyltaxol 0.0004 0.4 71.1 0.0 1.0 75.3Cephalomannine 10-deacetylbaccatin III 10-deacetyl-7-epitaxol 0.054 5.474.2 0.076 10.8 85.7 7-Epitaxol 0.009 0.9 74.6 0.009 1.3 86.2 UnknownTaxanes 0.203 20.5 79.7 0.240 34.1 90.2 Total Taxanes: 0.421 42.4 0.53375.8

TABLE 9 Nutrient medium manipulation for enhanced taxane and taxolbiosynthesis in Taxus chinensis suspension line K-1. 500 mg fresh weightcells were inoculated per 5 mL of medium and incubated in the dark for18 days. The total taxanes produced (in the cells and medium combined)is reported. The ingredients in media B & C are listed in Table 2.Medium B Medium C Taxane Level (mg/L) (mg/L) Baccatin III 4.3 3.97-xylosyl 10-deacetyl taxol 8.3 12.9 Cephalomannine 1.1 trace10-deacetyl 7-epi taxol 4.6 5.4 taxol 24.1 21.3 7-epi taxol 1.3 2.8other unidentified taxanes* 56.1 63.7 Total taxanes 99.8 mg/l 110 mg/l

TABLE 10 Enhancement of Taxane Biosynthesis in Taxus chinensis cell lineKS1A by Silver Dose mg/L extracellular product** Silver Compound (mM)Baccatin III Taxol Total Taxanes Culture Medium only* 16 5 21 Silverthiosulfate 50 71 15 86 Silver phosphate 100 48 7 55 Silver benzoate 2040 7 47 Silver sulfate 20 61 7 68 Toluenesulfonic acid silver 20 39 6 45salt Silver chloride 10 22 18 40 Silver oxide 50 43 18 61 Silver acetate10 52 10 62 Silver nitrate 20 63 6 69 *The culture medium was Medium Nfrom Table 2, with the addition of the following growth regulators: 10mM a-naphthaleneacetic acid, and 1 mM thidiazuron **All samples weretaken after 14 days of incubation.

TABLE 11 Enhancement of Taxol and Taxane Biosynthesis by Silver inseveral Taxus chinensis cell lines. The titers represent levels measuredin the whole broth, i.e., in the cells and in the extracellular medium.Other Total Cell Silver^(a) Culture Duration Baccatin III Taxol TaxanesTaxanes Culture Concentration Medium (days) mg/L mg/L mg/L (mg/L)SS6A-1224  0 I^(b) 30 10 48 23 81 SS6A-1224 50 mM I 30 172 86 126 384SS122-13  0 II^(c) 14 2 21 10 33 SS122-13 50 mM II 14 12 103 60 173SS122-42  0 II 14 3 80 26 109 SS122-42 50 mM II 14 4 146 38 188^(a)Added as silver thiosulfate ^(b)The culture medium is Medium N fromTable 2, with the addition of the growth regulator, a-naphthaleneaceticacid at a concentration of 10 mM. ^(c)The culture medium is Medium Nfrom Table 2, with the addition of the growth regulator,a-naphthaleneacetic acid at a concentration of 10 mM and thidiazuron ata concentration of 1 mM

TABLE 12 Enhancement of Taxol and Taxane Biosynthesis by Jasmonic acidand its methyl ester. Taxane titers were measured in the whole brothafter 14 days of cultivation. The culture medium was Medium N from Table2, with the additional presence of the growth regulator,a-naphthaleneacetic acid at a concentration of 10 mM. Other Total CellJasmonate Baccatin III Taxol Taxanes Taxanes Culture Concentration mg/Lmg/L mg/L (mg/L) SS122-42  0 3 80 26 109 SS122-42 200 mM JMA 4 120 87211 SS122-42  89 mM MJS 3 121 109 233 SS122-13  0 2 21 10 33 SS122-13 89 mM MJS 9 73 63 124 ^(a)JMA denotes the free acid, and MJS denotesmethyl jasmonate

TABLE 13 Enhancement of Taxol and Taxane Biosynthesis by3,4-methylenedioxy- nitrocinnamic acid (MDNA). Taxane levels weremeasured in the whole broth after 14 days of cultivation. The cell lineused was Taxus chinensis SS122-42. Other Total MDNA Culture Baccatin IIITaxol Taxanes Taxanes Concentration Medium^(a) mg/L mg/L mg/L (mg/L) 0 I3 80 26 109 50 mM I 5 163 45 213 50 mM II 34 311 89 434 ^(a)The culturemedium I refers to Medium N from Table 2, with the additional presenceof the growth regulator, a-naphthaleneacetic acid at a concentration of10 mM. The culture medium II is identical to Culture medium I, with theadditional presence of 50 mM silver thiosulfate.

TABLE 14 Enhancement of Taxol and taxanes in cell cultures of Taxuschinensis using various combinations of enhancement agents. All taxaneconcentrations are expressed as whole broth titers (i.e., concentrationin cells and medium combined), and values were obtained after 11 days ofincubation. Other Cell Culture Baccatin Taxol Taxanes Total TaxanesCulture Medium^(a) mg/L mg/L mg/L (mg/L) SS64-412 I 41 464 101 606SS64-561 II 590 182 388 1160 SS64-571 III 596 158 261 1015 SS124-77 IV72 39 576 687 SS122-29 V 18 306 152 476 SS85-26 VI 586 100 416 1102^(a)The culture medium for all combinations was Medium N in Table 2.Culture Medium I contained, in addition to Medium N, 10 mMa-naphthaleneacetic acid (NAA), 3 mM thidiazuron (TDZ), 50 mM3,4-methylenediox-6-ynitrocinnamic acid (MDNA), 89 mM methyl jasmonate(MJS), and 50 mM silver thiosulfate (SLTS). Culture Medium II contained,in addition Medium N, 10 mM NAA, 1 mM TDZ, 50 mM MDNA, 89 mM MJS, 10 mMSLTS, and an additional 98.5 mg/L sodium phosphate (monobasic). Culturemedium III contained, in addition to Medium N, 10 mM indolebutyric acid,3 μM TDZ, 30 mM 3,4-methylenediox-6-ycinnamic acid, 89 mM MJS, and 50 mMSLTS. Culture medium IV contained, in addition to Medium N, 10 mM NAA,89 mM MJS, 100 mM SLTS, and 5 mM glutamine. Culture medium V contained,in addition to Medium N, 10 mM NAA, 89 mM MJS, and 50 mM SLTS. Culturemedium VI contained, in addition to Medium N, 10 mM NAA, 1 mM TDZ, 50 mMMDNA, 18 mM MJS, 50 mM SLTS, and 5 mM glutamine.

TABLE 15 Enhancement of Taxane Production by Medium Exchange. Ave.Volumetric Culture Type of Duration Production Productivity^(e) CellLine Medium^(a) Operation^(b) (days) Product^(c) Level^(d) (mg/L)(mg/L/day) Paella I Batch 11 Taxol 185 13 Paella I Medium exchange 20Taxol 265 17 SS29-3A5 II Batch 14 Baccatin III 260 18 SS29-3A5 II Mediumexchange 28 Baccatin III 580 21 SS29-3A5 II Batch 22 10-deacetyl- 300 14baccatin III SS29-3A5 II Medium exchange 28 10-deacetyl- 400 14 baccatinIII SS45-146 III Batch 11 Total Taxanes 700 64 SS45-146 III Mediumexchange 28 Total Taxanes 2500 89 ^(a)The culture medium for theseculture conditions was Medium N in Table 2. Culture medium I included,in addition to Medium N, 10 mM a-naphthaleneacetic acid (NAA), 1 mMthidiazuron (TDZ), 50 mM 3,4-methylenedioxynitro-cinnamic acid (MDNA),18 mM methyl jasmonate (MJS), and 10 mM silver thiosulfate (SLTS).Culture medium II included, in addition to Medium N, 10 mM NAA, 1 mMTDZ, 50 mM MDNA, 89 mM MJS, 10 mM SLTS, and 5 mM glutamic acid(monopotassium salt). Culture medium III included, in addition to MediumN, 10 mM NAA, 1 mM TDZ, 50 mM MDNA, 89 mM MJS, 10 mM SLTS, and 5 mMglutamic acid ^(b)Repeated enhancement was achieved by medium exchange,as described in Example 14. ^(c)The predominant product produced by agiven cell line under the specified culture medium is listed; taxanesother than the predominant product were also produced in each case,except for cell line SS45-146, for which total taxane production islisted. ^(d)The production levels for batch cultivation refer toextracellular concentrations, i.e., the amount of taxane measured in theextracellular medium divided by the volume of the extracellular medium.For repeated enhancement by medium exchange, the production level refersto the total amount of taxane measured in the extracelluar medium aftereach medium exchange, divided by the suspension volume. ^(e)The averagevolumetric productivity is one indicator of biosynthetic capability; itis defined as the total product divided by the suspension volume, andfuther divided by the duration of the incubation.

TABLE 16.a Enhancement of Taxol and Taxane Production by Fed BatchOperation Total culture Baccatin Other Total Culture Type of Fed batchduration III Taxol taxanes taxanes Cell line medium^(a) operationcomponents^(b) (days) (mg/L)* (mg/L) (mg/L) (mg/L) CR-128 A Batch — 24152 134 203 489 A Fed batch F1 24 257 200 295 752 A Fed batch F2 24 254316 427 997 SS36-245 B Batch — 31 170 80 190 440 B Fed batch F3 31 50212 198 460 B Fed batch F4 31 56 412 348 816 SS36-359 C Batch — 21 220155 163 538 C Fed batch F5 21 439 182 304 925 ^(a)The culture medium forall cell lines was Medium N (Table 2). In addition, Culture medium Icontained 10 μM α-naphthaleneacetic acid (NAA), 30 μM3,4-methylenedioxy-6-nitrocinnamic acid (MDNA), 18 μM methyl jasmonate(MJS), and 50 μM silver thiosulfate (SLTS). Culture medium II contained,in addition to Medium N, 10 μM NAA, 50 μM MDNA, 50 μM SLTS, and 1 μMthidiazuron (TDZ). Culture medium III contained, in addition to MediumN, 10 μM NAA, 1 μM TDZ, 50 μM MDNA, 50 μM SLTS, 89 μM MJS. *All taxanevalues refer to whole broth titers: (mg taxanes in cells + mg taxanes inextracellular medium)/Total culture volume(liters).

TABLE 16.b Details of fed-batch operation described in Table 16.a.Duration Feed Feed rate Start of of feed solution Composition (mL/L/day)feed (day) (days) F1 25% (weight/volume) (w/v) fructose, 25 mM 10 7 17glutamine, 50 μM NAA, 250 μM SLTS, 89 μM MJS, 1.48 mM calcium chloride,0.63 mM magnesium sulfate, 0.68 mM sodium phosphate (monobasic). F2 F1,75 mM α-phenylalanine, 10 7 17 25 mM β-phenylalanine F3 25% (w/v)fructose, 10 6 25 150 mM α-phenylalanine, 25 mM β-phenylalanine F4 50%(w/v) glucose, 5.92 mM calcium chloride, 2.52 mM 5 9 22 magnesiumsulfate, 2.72 mM sodium phosphate (monobasic), 500 μM SLTS, 10 μM TDZ,100 μM NAA, 150 mM α-phenylalanine, 50 mM β-phenylalanine F5 contained50% (w/v) glucose, 5 12 9 100 μM NAA, 10 μM TDZ, 500 μM SLTS, 89 μM MJS,0.68 mM sodium phosphate (monobasic), 50 mM α-phenylalanine

TABLE 17 Enhancement of Taxol and taxanes in cell cultures of Taxuschinensis using various combinations of enhancement agents. All taxaneconcentrations are expressed as whole broth titers (i.e., concentrationin cells and medium combined). Other Total Cell Culture DurationBaccatin Taxol Taxanes Taxanes Culture Medium^(a) (days) mg/L mg/L mg/L(mg/L) SS122-41 I 20 106 374 158 638 SS122-41 I^(b) 20 7 507 148 662SS122-30 II 14 27 279 226 532 cr427 III 14 13 302 125 440 cr452 IV 14 11190 95 296 cr452 V 14 4 172 67 243 cr857 I 24 116 531 258 905 cr914 VI14 260 436 312 1008 ^(a)The culture medium for all combinations wasMedium N (Table 2) in which the primary carbon source was replaced byother sources as described in this legend. Culture Medium I contained100 g/l maltose instead of sucrose, and in addition, contained, 20 mM1-naphthaleneacetic acid (NAA), 40 mM 3,4-methylenedioxynitrocinnamicacid (MDNA), 45 mM methyl jasmonate (MJS), 100 mM silver thiosulfate(SLTS), and 5 mM glutamine. Culture Medium II contained 50 g/l maltoseinstead of sucrose, and in addition, contained, 10 mM NAA, 40 mM MDNA,100 mM MJS and 75 mM SLTS. Culture medium III contained 50 g/L maltoseinstead of sucrose, and in addition, contained, 20 mM NAA, 40 mM MDNA,45 mM MJS, 100 mM SLTS, and 5 mM glutamine. Culture medium IV contained50 g/l lactose instead of sucrose, and in addition, contained, 20 mMNAA, 40 mM MDNA, 45 mM MJS, 100 mM SLTS, and 5 mM glutamine. Culturemedium V contained 40 g/l galactose instead of sucrose, and in addition,contained, 20 mM NAA, 40 mM MDNA, 45 mM MJS, 100 mM SLTS, and 5 mMglutamine. Culture medium VI contained 70 g/L maltose instead of sucroseand in addition, contained, 20 mM NAA, 40 mM MDNA, 45 mM MJS, 100 mMSLTS, and 5 mM glutamine. ^(b)The fresh weight density was 26% (w/v).

TABLE 18.a Enhancement of Taxol and Taxane Production by Fed BatchOperation Baccatin Other Total Cell Culture Type of Fed batch III Taxoltaxanes taxanes culture medium^(c) operation components^(d) (mg/L)^(e)(mg/L) (mg/L) (mg/L) SS122-41^(a) A Batch — 120 225 123 468 A Fed batchF1 32 476 171 679 A Fed batch F2 27 501 180 708 SS122-41^(b) B Batch — 7507 148 662 B Fed batch F3 66 902 251 1219 ^(a)Inoculation density was20% (w/v) ^(b)Inoculation density was 26% (w/v) ^(c)The culture mediumfor all cell lines was Medium N (Table 2). The primary carbon source wassucrose unless substituted as described here. In addition, culturemedium A contained 20 μM α-naphthaleneacetic acid (NAA), 40 μM3,4-methylenedioxynitrocinnamic acid (MDNA), 45 μM methyl jasmonate(MJS), and 100 μM silver thiosulfate (SLTS), and 5 mM glutamine. Culturemedium B contained 100 mg/l maltose instead of sucrose, and in additioncontained, 20 μM NAA, 40 μM MDNA, 45 μM MJS, 100 μM SLTS, and 5 mMglutamine. ^(d)Refer to Table 18b ^(e)All taxane values refer to wholebroth titers: (mg taxanes in cells + mg taxanes in extracellularmedium)/Total culture volume(liters)

TABLE 18.b Details of fed-batch operation described in Table 18.a. StartDuration of of fed Feed Feed rate feed batch solution Composition(mL/L/day) (day) (days) F1 50% (weight/volume) (w/v) 8 10 11-21fructose, 50 mM glutamine F2 50% (w/v) maltose, 8 10 11-21 50 mMglutamine F3 50% (w/v) maltose, 200 μM 8 10 10-20 NAA, 450 μM MJS, 50 mMglutamine

1.-62. (canceled)
 63. A method for producing taxol and other taxanesfrom cell culture of a Taxus species comprising: cultivating insuspension culture, in a first medium developed for a growth phase andthen in a second medium developed for a product formation phase, cellsof a Taxus species derived from callus and/or suspension cultures, andrecovering said taxol and other taxanes from said cells, wherein saidstep of cultivating is carried out in the presence of 0.03% to 15% v/vof carbon dioxide in the gas phase in equilibrium with at least one ofsaid first medium and said second medium.
 64. The method of claim 63,wherein step of cultivating is carried out in the presence of 0.03% to15% v/v of carbon dioxide in the gas phase in equilibrium in said secondmedium.
 65. The method of claim 63, wherein step of cultivating iscarried out in the presence of 0.03% to 15% v/v of carbon dioxide in thegas phase in equilibrium in said first medium and in said second medium.66. The method of claim 63, wherein said step of cultivating is carriedout in the presence of 0.3% to 8% v/v of carbon dioxide in the gas phasein equilibrium with at least one of said first medium and said secondmedium.
 67. The method of claim 66, wherein step of cultivating iscarried out in the presence of 0.3% to 8% v/v of carbon dioxide in thegas phase in equilibrium in said second medium.
 68. The method of claim66, wherein step of cultivating is carried out in the presence of 0.3%to 8% v/v of carbon dioxide in the gas phase in equilibrium in saidfirst medium and in said second medium.
 69. The method of claim 63,wherein said Taxus species is Taxus chinensis.
 70. The method of claim64, wherein said Taxus species is Taxus chinensis.
 71. The method ofclaim 66, wherein said Taxus species is Taxus chinensis.
 72. The methodof claim 67, wherein said Taxus species is Taxus chinensis.
 73. Aprocess for recovering taxol and taxanes in high yield from cellcultures of a Taxus species, comprising: (a) culturing in one or morenutrient media, cells derived from a Taxus species under conditions toproduce taxol, said culturing comprising: (i) inoculating a Taxusspecies cells into growth and maintenance nutrient medium (1) insuspension to form an inoculated suspension; (ii) cultivating saidinoculated suspension of Step (i) to form a suspension culture; (iii)subculturing said suspension culture of Step (ii) into productionnutrient medium (2) to form said production culture; and (iv)cultivating said production culture of Step (iii) under conditions toproduce taxol and taxanes; and (b) recovering said taxol and taxanesfrom media, cells, or media and cells of said production culture of Step(iv), wherein said step of cultivating is carried out in the presence of0.03% to 15% v/v of carbon dioxide in the gas phase in equilibrium withat least one of said growth and maintenance nutrient medium (1) and saidproduction nutrient medium (2).
 74. The method of claim 73, wherein stepof cultivating is carried out in the presence of 0.03% to 15% v/v ofcarbon dioxide in the gas phase in equilibrium in said productionnutrient medium (2).
 75. The method of claim 73, wherein step ofcultivating is carried out in the presence of 0.03% to 15% v/v of carbondioxide in the gas phase in equilibrium in said growth and maintenancenutrient medium (1) and in said production nutrient medium (2).
 76. Themethod of claim 73, wherein said step of cultivating is carried out inthe presence of 0.3% to 8% v/v of carbon dioxide in the gas phase inequilibrium with at least one of said growth and maintenance nutrientmedium (1) and said production nutrient medium (2).
 77. The method ofclaim 76, wherein step of cultivating is carried out in the presence of0.3% to 8% v/v of carbon dioxide in the gas phase in equilibrium in saidproduction nutrient medium (2).
 78. The method of claim 76, wherein stepof cultivating is carried out in the presence of 0.3% to 8% v/v ofcarbon dioxide in the gas phase in equilibrium in said growth andmaintenance nutrient medium (1) and in said production nutrient medium(2).
 79. The method of claim 73, wherein said Taxus species is Taxuschinensis.
 80. The method of claim 74, wherein said Taxus species isTaxus chinensis.
 81. The method of claim 76, wherein said Taxus speciesis Taxus chinensis.
 82. The method of claim 77, wherein said Taxusspecies is Taxus chinensis.